Amidite derivatives and oligonucleotide derivatives

ABSTRACT

The present invention provides a compound of general formula (I): ##STR1## wherein X represents group (II) or (III): ##STR2## wherein Y represents a leaving group and Z represents an oligonucleotide. The compound can specifically transfer oligonucleotides to cells which specifically recognize a specified saccharide construction. Accordingly, the compound can be used as an antiviral agent or an antitumor agent.

This application is a 371 of PCT/JP96/00868 filed Mar. 29, 1996.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to amidite derivatives having amonosaccharide or a derivative thereof at their terminals. Inparticular, the present invention relates to oligonucleotide derivativesin which oligonucleotides are introduced into said amidite derivatives.

2. Description of the Related Art

In recent years, attempts have been made to suppress the expression oftargeted genes using oligonucleotides, specifically antisenseoligonucleotides. However, it was found that when administered directlyinto the body, the oligonucleotides were readily decomposed in theblood, or the greater portion was readily excreted in the urine.Moreover, the nucleotides were decomposed or excreted without beingincorporated into the targeted cells of lesioned organs.

To resolve these problems, it has been reported that the formation of aconjugate of asialoorosomucoid with poly-L-lysine yields a complex whichionically interacts with the antisense oligonucleotide of humanhepatitis B virus and the ionic complex enhanced the inhibitory effectof the antisence oligodeoxynucleotide on the biosynthesis of viralprotein significantly (G. Y. Wu and C. H. Wu (1992) J. Biol. Chem. 267,12436) and that the chloramphenicol acetyltransferase gene can betransferred into and expressed in the liver using a similar complex (G.Y. Wu and C. H. Wu (1991) Biotherapy 3, 879). Techniques used in thesereports are described in WO 93/04701 and 92/20316. Furthermore, it isreported in WO 93/19768 that a complex formed between DNA and asaccharide derivative, which was covalently coupled with a molecule tointercalate into DNA by inserting in the double helix structure (i.e.intercalator) was incorporated into a cell which specifically recognizedthe saccharide such that it was useful for the efficient expression ofgenetic information. Nakai et al intravenously injected an antisensenucleic acid complex, and a simulation of the amount delivered into theliver showed that this type of non-covalently bonded complex easilydissociates in the blood to preclude any significant transfer into theliver (D. Nakai, T. Seita and Y. Sugiyama (1995) Pharm. Tech. Japan, 11,27).

On the other hand, it is known that a compound in which galactose isintroduced into carboxymethylated dextran (M. Nishikawa et al. (1993)Pharmaceutical Research 10, 1253), and a compound in which galactose isintroduced into poly-L-glutamic acid are selectively distributed inhepatocytes (H. Hirabayashi et al (1994) Proceedings of the GeneralPresentation of the 144th Annual Meeting of Japan PharmacologicalAssociation 30(6) 15-4).

SUMMARY OF THE INVENTION

The present inventors have found that a complex of an oligonucleotidewith an amidite derivative having a monosaccharide residue at itsterminal is delivered to a specific organ and suppresses expression of aspecific gene in cells of the organ. The present invention is based onthese findings.

Accordingly, an objective of the present invention is to providecompounds which can incorporate oligonucleotides into organ cells,particularly into hepatocytes.

Another objective of the present invention is to provide amiditederivatives which are useful for synthesis of the compounds.

A compound of the present invention can be represented by generalformula (I): ##STR3## in which X is group (II): ##STR4## (in which Y isa leaving group) or group (III): ##STR5## (in which Z is anoligonucleotide or its derivative), T¹ is --(CH₂)s-- (in which srepresents an integer between 2 and 10), or (CH₂ CH₂ O)t--(CH₂)₂ -- (inwhich t represents an integer between 1 and 3),

T² is --(CH₂)u-- (in which u represents an integer between 2 and 10),--(CH₂ CH₂ O)v--(CH₂)₂ -- (in which v represents an integer between 1and 3), or group (IV): ##STR6## in which T¹ * and T¹ ** are each asdefined above for T¹, and n*, p*, q*, T³ *, T⁴ * and F³ are each asdefined below for n, p, q, T³, T⁴ and F¹, where each group and itsasterisk-labeled counterpart can be the same or different,

T³, T⁴ and T⁵, which may be the same or different, each represent--CONH--, --NHCO-- or --O--, provided that when either one of T³, T⁴ andT⁵ represents --O--, other two groups represent a group other than--O--,

F¹ and F², which may be the same or different, each represent amonosaccharide selected from the group consisting of galactose, glucoseand galactosamine, or a derivative thereof, or a disaccharide consistingof the monosaccharide and/or the derivative thereof, wherein a hydroxylgroup(s) which does not participate in any reactions in themonosaccharide, the derivative thereof and the disaccharide can beprotected,

m represents an integer between 0 and 10,

n represents an integer between 0 and 4,

p represents an integer between 0 and 4,

q represents an integer between 0 and 4 and

r represents an integer 0 or 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the inhibitory effect of the compounds of the presentinvention on expression of c-myc protein in HepG2 cells. Lanes 1, 2, 3and 4 are with compounds of Example 19 (1), Example 22, Example 21 andExample 19 (2), respectively. In all cases, the compounds were added ata concentration of 1.00 μM.

FIG. 2 shows the inhibitory effect of the compounds of the presentinvention on expression of c-my protein in HepG2 cells.

Lanes 1 and 8 are with no compound, Lanes 2-4 are with the compound ofExample 19 (2), and Lanes 5-7 are with the compound of Example 19 (1).The compounds were added at a concentration of 0.04 μM for the compoundsof Lanes 2 and 5, 0.20 μM for the compounds of Lanes 3 and 6 and 1 μMfor the compounds of Lanes 4 and 7.

FIG. 3 shows the effect of compounds of the present invention (Examples20 (1) and (2)) on down regulation of epidermal growth factor receptorsin a primary culture of hepatocytes isolated from rats.

FIG. 4 shows the effect of compounds of the present invention on growthof HepG2 cells. Black circles, black triangles, white circles and whitetriangles are with compounds of Example 19 (2), Example 19 (1), Example18 (2) and Example 18 (1), respectively.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Compounds of general formula (I)

In general formula (I), F¹, F² and F³ are a monosaccharide selected fromthe group consisting of galactose, glucose and galactosamine, preferablygalactose or galactosamine. The monosaccharide may be a derivativethereof. Examples of such derivative include an N- or O-acyl derivative(e.g., N-acetylgalactosamine), an O-alkyl derivative includingcarboxyalkyl derivatives (e.g., carboxymethyl derivatives), and an esterderivative with acids such as sulfuric acid, phosphoric acid andcarboxylic acid (e.g., sulfate ester derivatives), preferablyN-acetylgalactosamine.

F¹, F² and F³ may also be a disaccharide consisting of themonosaccharide and/or the monosaccharide derivative. Preferable examplesof such disaccharide include those having galactose, galactosamine orN-acetylgalactosamine at the non-reducible end, and are preferablylactose, lactosamine and N-acetyllactosamine.

In the present invention, the hydroxyl group of the monosaccharide andthe derivative thereof and the disaccharide which do not participate inany reactions can be protected. Examples of such protecting groupinclude an acyl group, preferably a straight chain or branched C₁₋₆(preferably C₁₋₄) alkylcarbonyl group, more preferably an acetyl group.Furthermore, in general formula (I), when group X is group (II), it ispreferable that non-reacting hydroxyl groups be protected, and whengroup X is group (III), it is preferable that non-reacting hydroxylgroups not be protected.

The monosaccharide and the derivative thereof and the disaccharideherein mean a saccharide in which one of the hydrogen atom(s) of thehydroxyl group(s) (preferably a hydroxyl group in an anomer position) inthe saccharide molecule is removed. In this case, bonds between F¹, F²and F³ and T¹, T² and T¹ ** can be either an α-glycosidic linkage or aβ-glycosidic linkage.

In T¹ and T², s and u are integers between 2 and 10, preferably between2 and 8, and t and v are integers between 1 and 3, preferably 2.

The compounds of the present invention may have a group (IV) describedabove in T² of general formula (I). The group represented by generalformula (IV) has substantially the same meaning as that represented bygeneral formula (I) without X--(CH₂)m--(T⁵)r-- and --F². Accordingly,T¹ * and T¹ ** are as defined in T¹, and may be the same as or differentfrom T¹. Furthermore, n*, p* and q* are integers in the range as definedin n, p and q and may be the same as or different from n, p and q.Furthermore, T³ *, T⁴ * and F³ are as defined in T³, T⁴ and F¹ and maybe the same as or different from T³, T⁴ and F¹.

In general formula (I), T³, T⁴ and T⁵, which may be the same ordifferent, each independently represent --CONH--, --NHCO-- or --O--,preferably --CONH--. Further, when one of T³, T⁴ and T⁵ is --O--, theremaining two are not --O--.

In general formula (I):

m is an integer between 0 and 10, preferably 0 or between 2 and 10, morepreferably between 3 and 9,

n is an integer between 0 and 4, preferably 0, 1 or 2, more preferably0,

p is an integer between 0 and 4, preferably 0, 1 or 2, more preferably0,

q is an integer between 0 and 4, preferably 1 or 2, more preferably 2,and

r is an integer 0 or 1, more preferably 1.

When r is 0, --(T⁵)r-- is a bond.

Examples of leaving groups represented by Y in group (II) include adiisopropylamino group and a morpholyl group (preferably morpholyl-4-ylgroup).

Examples of oligonucleotides represented by Z in group (III) include anoligodeoxyribonucleotide (DNA) and an oligoribonucleotide (RNA).Furthermore, their sequences and number of bases are not limited and canbe appropriately determined according to the use of the compounds.

Examples of nucleotide derivatives include those in which one or two ofthe oxygen atoms at a phosphoric ester bonding site are substituted byother atoms or groups as shown by the following formula: ##STR7##

Combinations of A¹ and A² and names of the resulting derivatives are asfollows:

                  TABLE 1                                                         ______________________________________                                        Combination of A.sup.1 and A.sup.2 and names of derivatives                        A.sup.1   A.sup.2    Name of derivative                                  ______________________________________                                        --OH       ═O     Phosphodiester (natural type)                             ═O --CH.sub.3 Methyl phosphonate                                          --OH ═S Phosphorothioate                                                  --SH ═S Phosphorodithioate                                                ═O --O--R Alkylphosphotriester                                            ═S --CH.sub.3 Methylphosphonothioate                                      ═O --NH--R Alkylphosphoramidite                                           ═O --BH.sub.3 Boranophosnate                                            ______________________________________                                    

In the table, R represents an alkyl group.

Further, substitution may occurred at all or a part of the phosphoricester bonds in the nucleotides and substitution may occurred at anatom(s) or group(s) in each phosphoric ester bond.

Examples of oligonucleotide derivatives which can be easily synthesizedinclude natural phosphodiesters and phosphorothioate derivatives.

Oligonucleotides represented by Z can be antisense oligonucleotides.Examples of antisense oligonucleotides include those which haveantiviral activity, in particular, an antisense oligonucleotide againstthe surface antigen of hepatitis B virus (HBsAg) (Goodarzi, G. at al(1990) J. Gen. Virol. 71, 3021) and an antisense oligonucleotide againstthe envelope protein of hepatitis B virus (HBeAg) (Blum, H. E. et al(1991) Lancet 337, 1230). Other examples include (2'-5')oligoadenylatewhich is known to be responsible for the antiviral activity ofinterferon and those which suppress expression of cancer genes.

Examples of oligonucleotides represented by Z include DNA sequencesshown in SEQ ID Nos: 1 to 3.

The sequence of SEQ ID No: 1 is a 15 mer oligodeoxynucleotide (sensesequence) having a sequence identical to the base sequence of 5 codonsstarting from the translation start codon toward the 3' end of messengerRNA derived from the human c-myc gene. The sequence of SEQ ID No: 2 is a15 mer oligodeoxynucleotide (antisense sequence) having a sequencecomplementary to the base sequence of 5 codons starting from thetranslation start codon toward the 3' end of messenger RNA derived fromthe human c-myc gene. The sequence of SEQ ID No: 3 is a 18 meroligodeoxynucleotide (antisense sequence) having a sequencecomplimentary to the sequence between the 33th and the 50th from the 3'end of messenger RNA derived from the rat epidermal growth factorreceptor protein.

A group of preferable compounds according to the present inventioninclude compounds of formula (I), in which

T¹ is --(CH₂)s-- (in which s represents an integer between 2 and 8) or--(CH₂ CH₂ O)₂ --(CH₂ )₂ --,

T² is --(CH₂)u-- (in which u represents an integer between 2 and 8),--(CH₂ CH₂ O)₂ --(CH₂ )₂ --, or group (IV) (in which T¹ * and T¹ ** areas defined for T¹ but each can be the same as or different from T¹, andn*, p*, q*, T³ *, T⁴ * and F³ are as defined thereinafter for n, p, q,T³, T⁴ and F¹, but can be the same as or different from n, p, q, T³, T⁴and F¹, respectively),

T³, T⁴ and T⁵ are --CONH--,

F¹ and F², which may be the same or different, each represent galactose,galactosamine, N-acetylgalactosamine, lactose, lactosamine orN-acetyllactosamine,

m is an integer 0 or between 2 and 10,

n is an integer 0, 1 or 2.

P is an integer 0, 1 or 2,

q is an integer 0, 1 or 2, and

r is an integer 1.

More preferable compounds according to the present invention arecompounds expressed by general formula (Ia): ##STR8## in which X isgroup (II): ##STR9## (in which Y is a leaving group) or group (III)##STR10## (in which z is an oligonucleotide or a derivative thereof) T¹is --(CH₂)s-- (in which s represents an integer between 2 and 8) or--(CH₂ CH₂ O)₂ --(CH₂)₂ --,

T² is --(CH₂)u-- (in which u represents an integer between 2 and 8),--(CH₂ CH₂ O)₂ --(CH₂)₂ --, or group (IVa): ##STR11## in which T¹ * andT¹ ** are as defined for T¹, and F³ is as defined thereinafter for F¹,but can be the same as or different from T¹ and F¹ respectively, and F¹and F², which may be the same or different, each represent amonosaccharide selected from the group consisting of galactose andgalactosamine, or a derivative thereof, or a disaccharide consisting ofthe monosaccharide and/or the derivative thereof, wherein a hydroxylgroup(s) which does not participate in any reactions in themonosaccharide, the derivative thereof and the disaccharide can beprotected, and

m is an integer between 3 and 9.

The compounds of the present invention have a monosaccharide or aderivative thereof at their terminals. Accordingly, the compounds of thepresent invention can deliver a specified sugar structure to cells whichspecifically recognize it.

Preparation of Compounds of General Formula (I)

The compounds of general formula (I) in which group X is not group (II)or group (III) but a hydroxyl group can be obtained by one of thefollowing method (1), (2) or (3): (1) A compound of formula (V):##STR12## (in which R¹ is a halogen atom, a protected or unprotectedhydroxyl group, amino group or carboxyl group, T¹⁻⁴, F¹, F², n, p and qare as defined above, but functional groups not involved in anyreactions of F¹ and F² are preferably protected)

may be reacted with the compound of formula (VI):

    R.sup.2 --(CH.sub.2)m--R.sup.1                             (VI)

(in which R¹ is as defined above, R² is a protected or unprotectedhydroxyl group, and m is as defined above) as follows: to form an amidebond, by a condensation method in the presence of a condensation agent(e.g., dicyclohexylcarbodiimide), by reaction with a mixed acidanhydride in the presence of isobutyl chlorocarbonate or the like, or byreaction with an active ester using hydroxysuccinimide or the like;alternatively to form an ether bond, by a condensation reaction betweena corresponding halogen compound and alkoxide. In either case, the usualreaction temperatures and reaction times for the respective method canbe applied.

The compound of formula (V) above can be obtained by reacting a compoundof formula: ##STR13## (in which R¹, n, p and q are as defined above)with a compound of formula (VIII):

    R.sup.1 --T.sup.1 --F.sup.1                                (VIII)

(in which R¹, T¹ and F¹ are as defined above) by a condensation reactionor the like as described above, followed by deprotection if necessary.Further, a compound of formula (V) in which T² is represented by group(IV) can be obtained by reacting (e.g., condensation) two of the same ortwo different compounds of formula (VII), occasionally followed bydeprotection if necessary, then by reacting the intermediate with acompound of formula (VIII).

(2) A compound of formula (IX): ##STR14## (in which R¹, R², T⁵, m, n, pand q are as defined above) and a compound of formula (VIII) are reactedby a condensation reaction or the like as described above, occasionallyfollowed by deprotection if necessary, to obtain the target compound.

The compound of formula (IX) can be obtained by reacting a compound offormula (VII) with a compound of formula (VI) by a condensation reactionor the like as described above, followed by deprotection if necessary.Further, a compound of formula (I) in which T² is represented by group(IV) can be obtained by reacting a compound of formula (VII) with acompound of formula (IX) by a condensation reaction or the like asdescribed above, followed by deprotection if necessary, and thenreacting the intermediate with a compound of formula (VIII).

(3) A compound of formula (X): ##STR15## (in which R¹⁻⁵, R², m, n, p, qand r are as defined above) and a compound of formula (XI):

    D--F*                                                      (XI)

(in which D is a halogen atom, an acyloxy group (e.g., acetoxy group) orCCl₃ C(═NH)O--, and F* is F¹ or F²) are glycosylated at a reactiontemperature between -20° C. and room temperature for 10 minutes to 24hours, followed by deprotection if necessary, to obtain the targetcompound.

The compound of formula (X) above can be obtained by reacting a compoundof formula (IX) with a compound of formula (XII):

    R.sup.3 --T.sup.1 --R.sup.2                                (XII)

(in which R², R³ and T¹ are as defined above) by a condensation reactionor the like as described above, followed by deprotection if necessary.

A compound of general formula (I) in which group X is group (II) can beobtained by reacting a compound of general formula (I) in which group Xis a hydroxyl group with a compound of formula (XIII): ##STR16## (inwhich Y is a leaving group) in the presence of an activating reagent(e.g., tetrazole) at a reaction temperature between -20° C. and roomtemperature for 10 minutes to several hours.

A compound of general formula (I) in which group X is group (III) can beobtained by reacting a compound of general formula (I) in which group Xis group (II) with a nucleotide using an ordinary DNA synthesis methodsuch as the β-cyanoethylphosphoramidite method.

In the β-cyanoethylphosphoramidite method, nucleotides are firstimmobilized on a solid phase, then coupled with an amidite monomer (inwhich hydroxyl groups not involved in bonding are preferably protected)activated by an activating agent such as tetrazole, oxidized with anoxidizing agent (e.g., an aqueous iodine solution), and cleaved from thesolid phase, and deprotection if necessary. Natural phosphodiester-typeoligonucleotides to be immobilized on a solid phase can be obtained inadvance by repeating this reaction.

Furthermore, phosphorothioate-type oligonucleotides can be synthesizedusing a reagent which can generate free sulphur atoms in an oxidationreaction (e.g., Beaucage reagent).

Furthermore, various phosphoric ester bonds can be formed using amiditesin which oxygen atoms at phosphoric acid sites are substituted byvarious functional groups. For example, a phosphorodithioate-typeoligonucleotide can be obtained by oxidizing with sulphur atoms using5'-dimethoxytrityldeoxynucleoside 3'-(dimethylamino)phosphorothioamidite (W. K. D. Bill et al (1989) J. Am. Chem. Soc. 111,2321). Furthermore, a methylphosphonate-type phosphoric ester bond canbe formed using 5'-methoxytrityldeoxynucleoside 3'-methylphosphonate andmesitylenesulfonyl-3-nitrotriazole (P. S. Miller et al (1983) NucleicAcid Res. 11, 6225). Furthermore, an ethylphosphotriester-typephosphoric ester bond can be formed using5'-dimethoxytrityldeoxynucleoside3'-O-ethyl-N,N-diisopropylphosphoramidite (K. A. Gallo et al (1986)Nucleic Acid Res. 14, 7405).

The compounds so synthesized are purified by partition chromatography(e.g., octadecyl silica gel column chromatography), ion-exchangechromatography (e.g., anion-exchange column chromatography), affinitychromatography (e.g., RCA lectin affinity chromatography) or the like.

Use of Compounds/Pharmaceutical Compositions

The compounds of the present invention have a monosaccharide or aderivative thereof at their terminals. Therefore, the compounds of thepresent invention can be delivered specifically to cells which recognizea specified sugar structure. Furthermore, the compounds of the presentinvention can have an oligonucleotide or a derivative thereof at theirterminals. This oligonucleotide can be one which can suppress expressionof a specified gene in cells of a targeted organ, for example, anantisense oligonucleotide. Accordingly, the compounds of the presentinvention can be used as therapeutic agents for various diseases.

The compounds of the present invention can deliver an antisenseoligonucleotide which is effective as an anti-viral agent to hepaticcells infected with viruses to enhance anti-viral activity. Furthermore,the compounds of the present invention can deliver an antisenseoligonucleotide which is effective as an anti-malignant tumor agent tocancerous hepatic cells to enhance anticancer activity.

Another aspect of the present invention is to provide pharmaceuticalcompositions comprising the compound of the present invention togetherwith pharmaceutically acceptable carriers.

Thus, the pharmaceutical compositions can be used as a therapeutic agentfor malignant tumors (e.g., a therapeutic agent for cancers), ananti-viral agent, an antirheumatic agent (e.g., an agent to suppressproduction of tumor necrosis factor), an anti-inflammatory agent, ananti-allergy agent or an immunosuppressive agent (e.g., an agent toinhibit migration of immunocompetent cells to inflammatory sites), anagent to improve circular functions (e.g., agents to inhibit growth ofvascular smooth cells associated with re-obstruction of coronaryvessels), an agent to improve endocrine functions (e.g., agents toinhibit abnormal hormone secretion), or a therapeutic agent for diseaseswhich are caused by abnormal expression or functional abnormality ofspecific proteins and of which symptoms can be improved by suppressingexpression of the proteins (e.g., an agent to suppress abnormalexpression of receptor proteins of cells).

If the pharmaceutical composition is a therapeutic agent for malignanttumors, Z in formula (I) can be an antisense oligonucleotide to suppressexpression of cancer genes. If the pharmaceutical composition is ananti-viral agent, Z in formula (I) can be an antisense oligonucleotidehaving antiviral activity.

A pharmaceutical composition of the present invention can beadministered to human and other animals either orally or non-orally(e.g., intravenous and intramuscular injection, and subcutaneous,rectal, endermic and nasal administration).

The compounds of the present invention can be prepared into suitabledosage form depending on their use, such as tablets, capsules, granules,powders, pills, grains and troches for oral administration, injectablesolutions for intravenous or intramuscular injections, formulations forrectal administration, oily suppositories and water-solublesuppositories. These various pharmaceutical preparations can be preparedby ordinary methods using customary excipients, bulking agents, binders,wetting agents, disintegrating agents, surfactants, lubricating agents,dispersing agents, buffering agents, preservatives, solubilizing agents,antiseptics, flavor/odor controlling agents, analgesic agents,stabilizers or the like. Examples of the nontoxic additives to be usedinclude lactose, fructose, glucose, starch, gelatin, magnesiumcarbonate, synthetic magnesium silicate, talc, magnesium stearate,methylcellulose, carboxymethyl cellulose or salts thereof, gum arabic,polyethylene glycol, syrup, vaseline, glycerin, ethanol, propyleneglycol, citric acid, sodium chloride, sodium sulfite and sodiumphosphate. If necessary, effective components other than the compoundsof the present invention can be added.

The particular dose for each individual patient is determined as afunction of usage, age and sex of the patient and severity of symptoms;however, a daily dose for an adult is generally between about 0.05 and250 mg, preferably between about 0.5 and 50 mg, which can beadministered as a single dose or divided into several doses.

In this specification, the term "therapy" means both the treatment andthe prevention of diseases.

Another aspect of the present invention is to provide a method fortreating a disease selected from the group consisting of a malignanttumor, a viral infection, an inflammatory disease, an allergic disease,an immune disease, a circulatory disease and an endocrine diseasecomprising administrating the compound of the present invention to ananimal (e.g., a mammal) including a human.

Another aspect of the present invention is to provide use of thecompound of the present invention for manufacturing a medicamentselected from the group consisting of a therapeutic agent for malignanttumors, an anti-viral agent, an antirheumatic agent, ananti-inflammatory agent, an anti-allergic agent, an immunosuppressiveagent, an agent to improve circulatory functions and an agent to improveendocrine functions, and use of the compound of the present inventionfor a medicament selected from the group consisting of a therapeuticagent for malignant tumors, an anti-viral agent, an antirheumatic agent,an anti-inflammatory agent, an anti-allergic agent, an immunosuppressiveagent, an agent to improve circulatory functions and an agent to improveendocrine functions.

EXAMPLES

The present invention will be explained by the following examples;however, the invention is not intended to be limited to these examples.

The following abbreviations are used: Boc: benzyloxycarbonyl group, THF:tetrahydrofuran, DMF: dimethylformamide, EDC:1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, DMAP:4-dimethylaminopyridine, TLC: thin layer chromatography, DMT group:dimethyltrityl group, TEAA: triethylammonium acetate, ODS column:octadecyl silica gel column.

Synthesis Example 1

Synthesis of Boc-L-glutamyl-L-glutamic acid α',α,γ-tribenzyl ester

15 g (45 mmol) of Boc-L-glutamic acid α-benzyl ester and 455 g (45 mmol)of N-methylmorpholine were mixed and dissolved in 300 ml of dry THF, andthe resulting solution was cooled and stirred on a dry ice/acetone bathunder a nitrogen gas flow. To this solution, a dry THF solutioncontaining 1 equivalent of ethyl chloroformate (4.89 g/10 ml) was addeddropwise for about 5 minutes, and the resulting reaction solution wasstirred at -30° C. for 1 minute. The solution was again cooled on a dryice/acetone bath and stirred, and a dry DMF solution containing 1equivalent of L-glutamic acid dibenzyl ester tosylate andN-methylmorpholine (22.5 g and 4.55 g/50 ml, respectively) were addeddropwise. The solution was stirred at a temperature between -40 and -20°C. for 1 hour and then at a temperature between -20 and 10° C. for 1hour to complete the reaction. Insoluble matter was removed byfiltration using Celite. After concentrating the solvents, theconcentrate was dissolved by adding ether (400 ml). The resulting ethersolution was washed consecutively with 5% citric acid, an aqueoussaturated sodium bicarbonate solution and an aqueous saturated sodiumchloride solution. After drying on magnesium sulphate and concentratingunder vacuum, the resulting residue was crystallized in isopropyl ether.Next, the resulting crystals were filtered and dried under vacuum toobtain 27.90 g of the title compound as a white powder.

Yield: 95.9%, mp=92-93° C. [α]_(D) =-25.8 (C=1.06, 24° C., methanol)

Synthesis Example 2

Synthesis of N-(10-hydroxydecanoyl)-L-glutamyl-L-glutamic acidα',α,γ-tribenzyl ester

(1) Synthesis of 10-hydroxysuccinimidyl decanoate active ester

7.53 g (40 mmol) of 10-hydroxydecanoic acid and 11.5 g (100 mmol) ofN-hydroxysuccinimide were dissolved in 80 ml of dry DMF, and 19.0 g (0.1mol) of EDC was added while stirring at room temperature. After stirringat room temperature overnight, the resulting reaction solution wasconcentrated under vacuum. Cold water was added to the concentratedresidue, and the admixture was stirred and then centrifuged. Theresulting precipitate was washed with cold water (150 ml×2) and thendissolved in chloroform. The resulting solution was dried on magnesiumsulphate and concentrated under vacuum. 7.57 g of 10-hydroxysuccinimidyldecanoate was obtained as a white powder.

Yield: 66.3%. NMR (500 MHz in CDCl₃): δ_(TMS) =1.24-1.46 (9H, m,--(CH₂)₄ --, and OH), 1.5-1.6 (4H, m, --(CH₂)₄ --), 1.75 (2H, quintet,J=7.5 Hz, --CH₂ --CH₂ --CO₂ Su), 2.60 (2H, t, J=7.5 Hz, --CH₂ --CO₂ Su),2.76-2.94 (4H, m, --(CH₂)₂ -- on Su), 3.64 (2H, t, J=6.5 Hz, --CH₂--OH); IR(KBr): νcm⁻¹ =3440(OH), 1820(COOSu), 1790, 1740, 1730

(2) Synthesis of N-(10-hydroxydecanoyl)-L-glutamyl-L-glutamic acid α',α,γ-tribenzyl ester

6.47 g (10 mmol) of the compound obtained in Synthesis Example 1 weredissolved in 50 ml of dry methylene chloride, and 15 ml oftrifluoroacetic acid were added while cooled in an ice-ethanol bath.After stirring at room temperature for 1 hour and concentrating undervacuum, the resulting residue was dissolved in an aqueous saturatedsodium bicarbonate-chloroform mixed solution (40 ml-100 ml). Afterextracting with chloroform, the organic layer was washed with an aqueoussaturated sodium chloride solution, dried on magnesium sulphate, andconcentrated under vacuum. 0.1 g of DMAP and dry acetonitrile (50 ml)was added immediately to dissolve the concentrated residue. Theadmixture was stirred while cooled in an ice bath, a dry acetonitrilesolution containing 4.28 g (15 mmol) of 10-hydroxysuccinimidyl decanoate(20 ml) was added, and the reaction was carried out overnight. 10 ml ofan aqueous saturated sodium bicarbonate solution were added. Theadmixture was stirred for 20 minutes and then concentrated under vacuum.The resulting residue was dissolved in 50 ml of chloroform and washedconsecutively with an aqueous saturated sodium bicarbonate solution (30ml) and an aqueous saturated sodium chloride solution (30 ml×2). Afterdrying on magnesium sulphate and concentrating under vacuum, theresulting residue was purified on silica gel column chromatography (150g, chloroform:ethyl acetate=4:1) to obtain 5.22 g of the title compoundas a white powder.

Yield: 71.8%, mp=86-88° C.; [α]_(D) =-22.2 (C=0.98, 25° C., methanol);NMR (500 MHz, in CDCl₃): δ_(TMS) =1.20-1.38 (10H brm, --(CH₂)₅ --, ondecanoic acid), 1.50-1.65 (7H, m, --(CH₂)₃ -- on decanoyl and OH),1.90-2.07 (2H, m, β-CH₂ on Glu×1/2×2), 2.12-2.28 (6H, m, β-CH₂ onGlu×1/2×2, --CH₂ --CO on dedanoyl and γ-CH₂ on Glu×1/2×2), 3.62 (2H, q,J=6.5 Hz, CH₂ OH), 5.04-5.22 (6H, m, PhCH₂ O×3), 6.48 (1H, d, J=7.5 Hz,NH on Glu), 6.52 (1H, d, J=7.5 Hz, NH on Glu), 7.24-7.42 (15H, m, C₆ H₅×3); IR(nujol): νcm⁻¹ =3290(OH), 1740(COOSu), 1640(CONHCO); FAB-MS:m⁺/z=717(M+H⁺)

Synthesis Example 3

Synthesis of N-(4-benzyloxybutynoyl)-L-glutamyl-L-glutamic acidα',α,γ-tribenzyl ester

(1) Synthesis of 4-benzyloxybutyric acid

4.0 g (60%, 0.1 mol) of sodium hydride were washed in hexane and mixedwith 30 ml of dry DMSO, and the resulting admixture was stirred at 60°C. on an oil bath for 1 hour under a nitrogen gas flow. The mixture wasstirred at room temperature, and dry DMSO containing 12.6 g (0.1 mol) ofsodium 4-hydroxybutyrate (100 ml) was added. After stirring at roomtemperature for 2 hours, the reaction solution was cooled on an icebath, 0.2 mol of benzyl bromide was added, and the reaction was carriedout at room temperature for 2 hours. After solidification, 0.5 L ofether was added, the admixture was filtered, the resulting residue waswashed with ether (300 ml×3), and the filtrate was concentrated undervacuum. The resulting residue was mixed with 100 ml of methanol, 100 mlof 8% sodium hydroxide were added, and the admixture was stirred at 60°C. for 15 hours. After drying under vacuum, the resulting concentratedresidue was washed with ether (200 ml×2), and the ether layer wasextracted with 1 N sodium hydroxide (50 ml). All aqueous layers werecombined, neutralized with concentrated hydrochloric acid (pH<4) andthen extracted with ether (100 ml×5). The ether layer was extracted with2 N sodium hydroxide (50 ml×3), all water layers were neutralized withconcentrated hydrochloric acid (pH<4), and further extracted with ether.The resulting product was washed with an aqueous saturated sodiumchloride solution, dried on magnesium sulphate, and then concentratedunder vacuum. 9.42 g of 4-benzyloxybutyric acid was obtained as a paleyellow oil.

Yield: 49.5%. NMR (500 MHz, in CDCl₃): δ_(TMS) =1.95 (2H, dt, J=6 and 7Hz, β-CH₂), 2.50 (2H, t, J=7 Hz, α-CH₂), 3.54 (2H, t, J=6 Hz, γ-CH₂),4.52 (2H, s, PhCH₂ O), 7.25-7.4 (5H, m, C₆ H₅), 10.0-10.8 (1H, br, CO₂H); IR (neat): νcm⁻¹ =1710(COOH); FAB-MS:m⁺ /z=195(M+H⁺)

(2) Synthesis of N-(4-benzyloxybutyroyl)-L-glutamyl-L-glutamic acidα',α,γ-tribenzyl ester

6.47 g (10 mmol) of the compound obtained in Synthesis Example 1 weredissolved in 50 ml of dry methylene chloride, and 15 ml oftrifluoroacetic acid were added while cooled in an ice-ethanol bath.After stirring at room temperature for 1 hour and concentrating undervacuum, the resulting residue was dissolved in an aqueous saturatedsodium bicarbonate-chloroform mixed solution (40 ml-100 ml). Afterextracting with chloroform, the resulting organic layer was washed withan aqueous saturated sodium chloride solution, dried on magnesiumsulphate, and concentrated under vacuum. 2.91 g (15 mmol) of4-benzyloxybutyric acid, 0.1 g of DMAP and dry acetonitrile (50 ml) wereadded immediately to dissolve the resulting residue. The admixture wasstirred while cooled in an ice bath, a dry acetonitrile solution (10 ml)containing 3.1 g (15 mmol) of DCC was added, and the reaction wascarried out for 16 hours. After drying under vacuum, the resultingconcentrated residue was dissolved in 150 ml of chloroform and washedconsecutively with 5% citric acid (30 ml×2), an aqueous saturated sodiumbicarbonate solution (30 ml) and an aqueous saturated sodium chloridesolution (30 ml×2). After drying on magnesium sulphate and concentratingunder vacuum, the resulting residue was purified on silica gel columnchromatography (180 g, hexane:chloroform:ethyl acetate=1:1:1) to obtain6.065 g of the title compound as a white powder.

Yield: 83.9%, mp=100-105° C. [α]_(D) =-20.7 (C=1.03, 24° C., methanol)NMR (500 MHz, in CDCl₃): δ_(TMS) =1.85-2.48 (16H, m, β-CH₂ on Glu×2,γ-CH₂ on Glu×2, and BnO--CH₂ --CH₂ --CH₂ --CO), 3.49 (2H, t, J=6 Hz,BnO--CH₂), 4.47 (1H, d, J=12 Hz, PhCH₂ O×1/2), 4.49 (1H, d, J=12 Hz,PhCH₂ O×1/2), 4.54-4.64 (2H, m, α-CH on Glu×2), 5.07 (1H, d, J=12 Hz,PhCH₂ OCO×1/2), 5.08 (1H, d, J=12 Hz, PhCH₂ OCO×1/2), 5.12 (1H, d, J=12Hz, PhCH₂ OCO×1/2), 5.15 (2H, s, PhCH₂ OCO×1/2), 5.16 (1H, d, J=12 Hz,PhCH₂ OCO), 6.49 (1H, d, J=7.5 Hz, NH), 6.59 (1H, d, J=7.5 Hz, NH),7.20-7.40 (20H, m, C₆ H₅ ×4), 10.0-10.8 (1H, br, CO₂ H); IR (nujol):νcm⁻¹ =3300(NH), 1740(COOBn), 1725(COOBn), 1650(CONH), 1645(CONH);FAB-MS:m⁺ /z=723(M+H⁺)

Example 1

Synthesis of N-benzyloxycarbonyl-L-glutamic acidα,γ-di-2--(2',3',4',6'-tetraacetyl-β-D-galactosyl-1')ethoxyethoxyethylamide

To 0.506 g (1.80×10⁻³ mol) of N-benzyloxycarbonyl-L-glutamic acid wereadded 0.539 g (1.3 equivalent, molar ratio=2.6) of N-hydroxysuccinimideand 15 ml of acetonitrile, and the resulting solution was stirred whilecooled on ice. To this solution, 0.817 g (1.1 equivalent, molarratio=2.2) of N,N-dicyclohexyl-carbodiimide was added, and the resultingreaction mixture was stirred at 14° C. for 14 hours.

Separately, to 2.388 g (1.0 equivalent, mol ratio 2.0) of1-(2'-azidoethoxyethoxyethyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranosewere added 10 ml of acetonitrile, and the resulting solution was cooledon ice. To this solution, 403 μl [1.0 equivalent to1--(2'-azidoethoxyethoxyethyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranose]of N-methylmorpholine were added, the resulting solution was added tothe abovementioned reaction mixture and the admixture was stirred at 4°C. for 18 hours. The precipitate was removed by filtration and thesolvents were removed by distillation under vacuum. The residue thusobtained was dissolved in ethyl acetate and washed with water/an aqueoussaturated sodium chloride solution (1/1), dried on magnesium sulphate,and the solvents were removed by reduced-pressure distillation. Theresidue was purified by chromatography on silica gel eluting withbenzene:acetone=2:3 to obtain 1.366 g of the title compound as anamorphous colorless solid.

Yield: 63.0%. ¹ H-NMR (δ, CDCl₃): 1.99 (s, 6H, acetyl), 2.04-2.07 (m,2H, Gluβ), 2.05 (s, 6H, acetyl), 2.05 (s, 3H, acetyl), 2.06 (s, 3H,acetyl), 2.15 (s, 6H, acetyl), 2.25-2.30 (m, 1H, Gluγ), 2.33-2.39 (m,1H, Gluγ), 3.40-3.75 (m, 22H, ethyleneglycol moiety), 3.90-3.98 (m, 4H,GalClβ-OCH₂ CH₂ O-- and Gal C5-H), 4.10-4.14 (m, 2H, Gal C6-Ha),4.16-4.22 (dd, 3H, Gal C6-Hb and Gluα), 4.54 (d, 1H, J1.2=7.8 Hz, GalCl-H), 4.55 (d, 1H, J1.2=7.8 Hz, GalCl-H), 5.01-5.04 (m, 2H, Gal C3-H),5.09 (s, 2H, PhCH₂ O--), 5.18-5.22 (m, 2H, Gal C2-H), 5.39 (br d, 2H,Gal C4-H), 6.06 (d, 1H, J=6.8 Hz, ZNH--), 6.64 (br s, 1H, --CONH--),7.16 (br s, 1H, --CONH--), 7.29-7.38 (m, 5H, Ph(Z)); IR (KBr tab): 1751cm⁻¹ (C═O); [α]_(D) ²⁴ =-8.6 (c=0.97, CHCl₃)

Example 2

Synthesis of N-t-butoxycarbonyl-γ-L-glutamyl-L-glutamic acidα',α,γ-tri-2-(2',3',4',6'-tetraacetyl-β-D-galactosyl-1')ethoxyethoxyethylamide

To 1.061 g (3.59×10⁻³ mol) of N-t-butoxycarbonyl-L-glutamyl-L-glutamicacid (12) were added 1.486 g (1.2 equivalent, molar ratio=3.6) ofN-hydroxysuccinimide and 50 ml of acetonitrile, and the resultingsolution was stirred while cooled on ice. To this solution, 2.442 g (1.1equivalent, molar ratio=3.3) of N,N-dicyclohexylcarbodiimide were added,and the resulting reaction mixture was stirred at 4° C. for 27 hours.

Separately, to 6.962 g (1.03 equivalent, mol ratio 3.09) of1--(2'-azidoethoxyethoxyethyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranosewere added 10 ml of acetonitrile, and the resulting solution was cooledon ice. To this solution, 1.17 ml [1.0 equivalent to1-(2'-azidoethoxyethoxyethyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranose]of N-methylmorpholine were added, the resulting solution was added tothe abovementioned reaction mixture and the admixture was stirred at 4°C. for 18 hours. The precipitate was removed by filtration and thesolvents were removed by distillation under vacuum. The residue thusobtained was dissolved in ethyl acetate and washed with water/an aqueoussaturated sodium chloride solution (1/1), dried on magnesium sulphate,and the solvents were removed by reduced-pressure distillation. Theresidue was purified by chromatography on silica gel eluting withchloroform-methanol=20:1 to obtain 4.700 g of the title compound as anamorphous colorless solid.

Yield: 77.4%. ¹ H-NMR(δ, CDCl₃): 1.42 (s, 9H, t-Bu), 1.94-2.20 (m, 4H,Gluβ), 1.99 (s, 9H, acetyl), 2.05-2.06 (m, 18H, acetyl), 2.16 (s, 9H,acetyl), 2.25-2.40 (m, 4H, Gluγ), 3.32-3.76 (m, 33H, ethyleneglycolmoiety), 3.91-4.00 (m, 6H, GalClβ-OCH₂ CH₂ O-- and Gal C5-H), 4.10⁻⁴.15(m, 4H, Gal C6-Ha and Boc-Gluα), 4.16-4.20 (dd, 3H, Gal C6-Hb), 4.38(brdd, 1H, Glu (Gluα)), 4.55-4.57 (m, 3H, Gal Cl-H), 5.02-5.05 (m, 3H, GalC3-H), 5.17-5.22 (m, 3H, Gal C2-H), 5.39 (br d, 3H, Gal C4-H), 5.47 (d,1H, BocNH--), 6.93(br s, 1H, --CONH--), 7.18-7.26 (m, 2H, --CONH--),7.68 (br s, 1H, --CONH--)

Example 3

Synthesis of N-t-butoxycarbonyl-γ-L-glutamyl-L-glutamic acidα',α,γ-tri-2-(2',3',4',6'-tetraacetyl-β-D-glucosyl-1')ethoxyethoxyethylamide

The title compound was obtained as an amorphous pale brown solid in thesame manner as described in Example 2, except that1-(2'-azidoethoxyethoxyethyl)-2,3,4,6-tetra-O-acetyl-β-D-glucopyranosewas used instead of1-(2'-azidoethoxyethoxyethyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranose.

Yield: 67.0%. ¹ H-NMR (δ, CDCl₃):

1.42 (s, 9H, t-Bu), 1.92-2.16 (m, 4H, Gluβ), 2.01 (s, 9H, acetyl), 2.03(s, 9H, acetyl), 2.05 (s, 6H, acetyl), 2.05 (s, 3H, acetyl), 2.09 (s,9H, acetyl), 2.24-2.44 (m, 4H, Gluγ), 3.32-3.76 (m, 33H,ethyleneglycolmoiety), 3.92-3.98 (m, 3H, Glc Clβ-OCH₂ CH₂ O--), 4.11 (brq, 1H, Boc-Gluα), 4.15 (dd, 1H, J5.6a=2.0 Hz, J6a, 6b=12.2 Hz,GlcC6-Ha), 4.27 (dd, 1H, J5.6a=4.8 Hz, J6a, 6b=12.2 Hz, Glc C6-Hb), 4.38(br q, Glu(Gluα)), 4.59-4.60 (m, 3H, Glc Cl-H), 4.99 (br t, 3H, GlcC2-H), 5.09 (br t, 3H, Glc C4-H), 5.21 (br t, 3H, Glc C3-H), 5.45 (br d,1H, BocNH--), 6.87 (br s, 1H, --CONH--), 7.19 (br s, 2H, --CONH-- andGluγ--CONH), 7.06 (br s, 1H, --CONH--) IR(KBr tab): 1757 cm⁻¹ (C=O)

Example 4

Synthesis of N-(10-hydroxydecanoyl)-L-glutamyl-L-glutamic acidα',α,γ-tri-2--(2',3',4',6'-tetraacetyl-β-D-galactosyl-1)hexylamide

2.31 g (4.8 mmol) of1-(6'-azidohexyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranose weredissolved in 50 ml of ethanol, and 750 mg of methanesulfonic acid and 2g of Lindlar catalyst were added. The admixture was stirred under ahydrogen gas flow under pressure (50 psi) for 2 hours. After addinganother 1 g of Lindlar catalyst, the admixture was again stirred under ahydrogen gas flow under pressure (50 psi) for 1 hour. The catalyst wasremoved by filtration, the filtrate was concentrated under vacuum, andthe resulting residue was dissolved in 50 ml of dry acetonitrile toobtain an amine solution.

1.1 g (1.5 mmol) of the compound of Synthesis Example 2 were dissolvedin a dioxane-water mixture (30 ml-10 ml), and 300 mg of 10%palladium-carbon catalyst were added. The admixture was stirred under ahydrogen gas flow under normal pressure for 20 hours. The catalyst wasremoved by filtration, the filtrate was concentrated under vacuum, andthe resulting residue was dissolved in a dry acetonitrile-DMF mixture(20 ml-5 ml). 1.15 g (10 mmol) of N-hydroxysuccinimide were added, andthe admixture was stirred while cooled in an ice-ethanol bath. 1.03 g ofDCC were added, and the reaction was carried out at 0-5° C. for 4 hours.The precipitate was removed by filtration and washed with a small amountof acetonitrile to obtain an active ester solution.

The amine solution (6 mmol) and the active ester solution (1.5 mmol)were mixed while cooled on an ice bath, 10 mmol of diisopropylethylaminewas added, and the reaction was carried out at 4° C. overnight. Afterconcentration under vacuum, the resulting residue was dissolved in 150ml of ethyl acetate, and washed consecutively with 5% citric acid (15ml×4), an aqueous saturated sodium bicarbonate solution (15 ml×6) and anaqueous saturated sodium chloride solution (15 ml×2). After drying onmagnesium sulphate, the residue was concentrated under vacuum andpurified by chromatography on silica gel (150 g, eluting withchloroform:ethanol=30:1) to obtain 744 mg of the title compound as anamorphous white powder.

Yield: 28.6%. [α]_(D) =-11.3 (C=1.00, 26° C., methanol) NMR (500 MHz, inCDCl₃): δ^(TMS) =1.24-1.42 (22H, brm, --(CH₂)₅ -- on --(CH₂)₉ -- and--(CH₂)₂ -- on --(CH₂)₆ --×3), 1.42-1.68 (16H, brm, --(CH₂)--×8),1.8-2.45, 2.45-2.57,3.01 (46H, m, CH₂ CO on decanoyl, CH₃ CO×12, β-CH₂on Glu×2, and γ-CH₂ on Glu×2), 3.1-3.25 (6H, m, NCH₂ ×3), 3.42-3.53 (3H,m, O-CH₂ ×1/2×3), 3.64 (2H, m, changed with D20, HOCH₂), 3.83-3.95 (6H,m, 5-CH on Gal×3 and O--CH₂ ×1/2×3), 4.08-4.24 (6H, m, 6'-CH₂ on Gal×3),4.38 (1H, m, α-CH on Glu), 4.46 (3H, d, J=8.5 Hz, 1'-CH on Gal×3), 4.64(1H, m, α-CH on Glu), 5.00-5.06 (3H, m, 3'-CH on Gal×3), 5.19 (3H, m,2'-CH on Gal×3), 5.39 (3H, m, 4'-CH on Gal×3), 6.25 (1H, t, J=6 Hz,NH--CH₂), 6.36 (1H, d, J=8 Hz, NH--CH), 6.63 (1H, t, J=6 Hz, NH--CH₂),6.72 (1H, d, J=7 Hz, NH--CH), 7.06 (1H, t, J=6 Hz, NH--CH₂), 7.21 (1H,m, disappeared with D₂ O, OH); IR(nujol): νcm⁻¹ =3290(OH and CONH),1750(CH₃ CO); FAB-MS: m⁺ /z=1734(M+H⁺)

Example 5

Synthesis of N-(10-hydroxydecanoyl)-L-glutamyl-L-glutamic acidα',α,γ-tri-2-(2',3',4',6'-tetraacetyl-β-D-galactosyl-1)ethylamide

2.50 g (6 mmol) of1-(2'-azidoethyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranose weredissolved in 40 ml of ethanol, and 750 mg of methanesulfonic acid and 2g of Lindlar catalyst were added. The admixture was stirred under ahydrogen gas flow under pressure (50 psi) for 2 hours. After addinganother 1 g of Lindlar catalyst, the admixture was again stirred under ahydrogen gas atmosphere under pressure (50 psi) for 1 hour. The catalystwas removed by filtration, the filtrate was concentrated under vacuum,and the resulting residue was dissolved in 50 ml of dry acetonitrile toobtain an amine solution.

1.1 g (1.5 mmol) of the compound of Synthesis Example 2 were dissolvedin a dioxane-water mixture (30 ml-10 ml), and 300 mg of 10%palladium-carbon catalyst were added. The admixture was stirred under ahydrogen gas atmosphere under normal pressure for 20 hours. The catalystwas removed by filtration, the filtrate was concentrated under vacuum,and the resulting residue was dissolved in a dry acetonitrile-DMFmixture (20 ml-5 ml). 1.15 g (10 mmol) of N-hydroxysuccinimide wereadded, and the admixture was stirred while cooled in an ice-ethanolbath. 1.03 g of DCC were added, and the reaction was carried out at 0-5°C. for 4 hours. The precipitate was removed by filtration and washedwith a small amount of acetonitrile to obtain an active ester solution.

The amine solution (6 mmol) and the active ester solution (1.5 mmol)were mixed while cooled on an ice bath, 10 mmol of diisopropylethylaminewas added, and the reaction was carried out at 4° C. overnight. Afterconcentration under vacuum, the resulting residue was dissolved in 150ml of ethyl acetate, and washed consecutively with 5% citric acid (15ml×4), an aqueous saturated sodium bicarbonate solution (15 ml×6) and anaqueous saturated sodium chloride solution (15 ml×2). After drying onmagnesium sulphate, the residue was concentrated under vacuum andpurified by chromatography on silica gel (150 g, eluting withchloroform:ethanol=30:1→20:1) to obtain 1.33 g of the title compound asan amorphous white powder.

Yield: 56.6%. [α]_(D) =-8.9 (C=1.04, 24° C., methanol) NMR (500 MHz, inCDCl₃): δ^(TMS) =1.25-1.4 (10H brm, --(CH₂)₅ --), 1.52-1.64 (4H, m,--(CH₂)₂ --), 1.75 (1H, t, J=5 Hz, disappeared with D₂ O, OH), 1.9-2.25(42H, CH₂ CO on decanoyl, CH₃ CO×12 and β-CH₂ on Glu×2), 2.30-2.42 (4H,m, γ-CH₂ on Glu×2),3.36-3.52 (6H, brm, NCH₂ ×3), 3.60-3.74 (5H, m,changed with D₂ O, HOCH₂ and O--CH₂ ×1/2×3), 3.84-3.98 (6H, m, 5'-CH onGal×3 and O--CH₂ ×1/2×3), 4.08-4.22 (6H, m, 6'-CH₂ on Gal×3), 4.40-4.58(5H, m, α-CH on Glu×2 and 1'-CH on Gal×3), 5.00-5.07 (3H, m, 3'-CH onGal×3), 5.12-5.20 (3H, m, 2'-CH on Gal×3), 5.36-5.44 (3H, m, 4'-CH onGal×3), 6.61 (1H, d, J=7.5 Hz, NH--CH), 6.93 (1H, t, J=6 Hz, NH--CH₂),6.96 (1H, t, J=6 Hz, NH--CH₂), 7.15 (1H, d, J=7.5 Hz, NH--CH), 7.68 (1H,t, J=6 Hz, NH--CH); IR(nujol): νcm⁻¹ =3280(OH of CONH), 1750(CH₃ CO),1635(CONH); FAB-MS:m⁺ /z=1566(M+H⁺)

Example 6

Synthesis of N-(4-hydroxybutyroyl)-L-glutamyl-L-glutamic acidα',α,γ-tri-2-(2',3',4',6'-tetraacetyl-β-D-galactosyl-1)ethylamide

1.88 g (4.5 mmol) of1-(2'-azidoethyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranose weredissolved in 40 ml of ethanol, and 580 mg of methanesulfonic acid and 2g of Lindlar catalyst were added. The admixture was stirred under ahydrogen gas flow under pressure (50 psi) for 2 hours. After addinganother 1 g of Lindlar catalyst, the admixture was again stirred under ahydrogen gas atmosphere under pressure (50 psi) for 1 hour. The catalystwas removed by filtration, the filtrate was concentrated under vacuum,and the resulting residue was dissolved in 50 ml of dry acetonitrile toobtain an amine solution.

1.45 g (2 mmol) of the compound of Synthesis Example 3 were dissolved ina dioxane-water mixture (25 ml-10 ml), and 400 mg of 10%palladium-carbon catalyst were added. The admixture was stirred under ahydrogen gas flow under normal pressure for 18 hours. The catalyst wasremoved by filtration, the filtrate was concentrated under vacuum, andthe resulting residue was dissolved in a dry acetonitrile-DMF mixture(30 ml-10 ml). 1.73 g (15 mmol) of N-hydroxysuccinimide were added, andthe admixture was stirred while cooled in an ice-ethanol bath. 1.49 g ofDCC were added, and the reaction was carried out for 5 hours. Theprecipitate was removed by filtration and washed with a small amount ofacetonitrile to obtain an active ester solution. A half portion of thesolution was used for the next reaction.

The amine solution (4.5 mmol) and the active ester solution (1 mmol)were mixed while cooled on an ice bath, 2 ml of diisopropylethylaminewas added, and the reaction was carried out at 4° C. overnight. Afterconcentration under vacuum, the resulting residue was dissolved in 80 mlof ethyl acetate, and washed consecutively with 5% citric acid (10 ml),an aqueous saturated sodium chloride solution (5 ml), an aqueoussaturated sodium bicarbonate solution (10 ml×2) and an aqueous saturatedsodium chloride solution (10 ml×2). After drying on magnesium sulphate,the residue was concentrated under vacuum and purified by chromatographyon silica gel (100 g, eluting with chloroform:ethanol=15:1) to obtain622 mg of the title compound as an amorphous white powder.

Yield: 42.3%. [α]_(D) =-11.9 (C=1.025, 26° C., methanol) NMR (500 MHz,in CDCl₃): δ^(TMS) =1.89 (2H, quintet, J=6.5 Hz, CH₂ --CH₂ --OH),1.93-2.22 (40H, m, CH₃ CO×12 and β-CH₂ on Glu×2), 2.30-2.43 (6H, m,γ-CH₂ on Glu×2 and CH₂ --CH₂ --CH₂ --OH), 2.99 (1H, brm, disappearedwith D₂ O, OH), 3.36-3.55 (6H, m, NHC₂ ×3), 3.65-3.74 (5H, m, O--CH₂×1/2×3 and CH₂ OH), 3.85-3.99 (6H, m, 5'-CH on Gal×3 and O--CH₂ ×1/2×3),4.08-4.22 (6H, m, 6'-CH₂ on Gal×3), 4.37-4.50 (2H, m, α-CH on Glu×2),4.48-4.56 (3H, m, 1'-CH on Gal×3), 5.01-5.06 (3H, m, 3'-CH on Gal×3),5.12-5.18 (3H, m, 2'-CH on Gal×3), 5.40 (3H, d, J=3.5 Hz, 4'-CH onGal×3), 6.80 (1H, t, J=5.5 Hz, NH--CH₂), 6.88 (1H, d, J=7.5 Hz, NH--CH),6.93 (1H, t, J=5.5 Hz, NH--CH₂), 7.13 (1H, d, J=7 Hz, NH--CH), 7.54 (1H,t, J=7.5 Hz, NH--CH) IR(nujol): νcm⁻¹ =3280(OH of CONH), 1750(CH₃ CO),1635(CONH); FAB-MS:m⁺ /z=1482(M+H⁺)

Example 7

Synthesis of N-(4-hydroxybutynoyl)-L-glutamyl-L-glutamic acidα',{α,γ-tri-2-(2',3',4',6'-tetraacetyl-β-D-galactosyl-1)ethoxy}ethoxyethylamide

2.27 g (4.5 mmol) of1-(2'-azidoethoxyethoxyethyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranosewere dissolved in 40 ml of ethanol, and 580 mg of methanesulfonic acidand 2 g of Lindlar catalyst were added. The admixture was stirred undera hydrogen gas atmosphere under pressure (50 psi) for 2 hours. Afteradding another 1 g of Lindlar catalyst, the admixture was again stirredunder a hydrogen gas atmosphere under pressure (50 psi) for 1 hour. Thecatalyst was removed by filtration, the filtrate was concentrated undervacuum, and the resulting residue was dissolved in 50 ml of dryacetonitrile to obtain an amine solution.

1.45 g (2 mmol) of the compound of Synthesis Example 3 were dissolved ina dioxane-water mixture (25 ml-10 ml), and 400 mg of 10%palladium-carbon catalyst were added. The admixture was stirred under ahydrogen gas atmosphere under normal pressure for 18 hours. The catalystwas removed by filtration, the filtrate was concentrated under vacuum,and the resulting residue was dissolved in a dry acetonitrile-DMFmixture (30 ml-10 ml). 1.73 g (15 mmol) of N-hydroxysuccinimide wereadded, and the admixture was stirred while cooled in an ice-ethanolbath. 1.49 g of DCC were added, and the reaction was carried out for 5hours. The precipitate was removed by filtration and washed with a smallamount of acetonitrile to obtain an active ester solution. A halfportion of the solution was used for the next reaction.

The amine solution (4.5 mmol) and the active ester solution (1 mmol)were mixed while cooled on an ice bath, 2 ml of diisopropylethylaminewas added, and the reaction was carried out at 4° C. overnight. Afterconcentration under vacuum, the resulting residue was dissolved in 80 mlof ethyl acetate, and washed consecutively with 5% citric acid (10 ml),an aqueous saturated sodium chloride solution (5 ml), an aqueoussaturated sodium bicarbonate solution (10 ml×2) and an aqueous saturatedsodium chloride solution (10 ml×2). After drying on magnesium sulphate,the residue was concentrated under vacuum and purified by chromatographyon silica gel (100 g, eluting with chloroform:ethanol=10:1) to obtain697 mg of the title compound as a colorless caramel-like solid.

Yield: 43.5%. [α]_(D) =-11.0 (C=1.00, 26° C., metanol) NMR (500 MHz, inCDCl₃): δ^(TMS) =1.82-1.94 (2H, m, CH₂ --CH₂ --OH), 1.92-2.20 (40H, m,CH₃ CO×12 and β-CH₂ on Glu×2), 2.27-2.44 (6H, m, γ-CH₂ on Glu×2 and CH₂--CH₂ --CH₂ --OH), 3.21 (1H, t, J=5 Hz, disappeared with D₂ O, OH),3.33-3.76 (35H, m, NCH₂ ×3, OCH₂ ×12, O--CH₂ -Gal×1/2×3 and CH₂ OH),3.92-4.00 (6H, m, 5'-CH on Gal×3 and O--CH₂ ×1/2×3), 4.08-4.22 (6H, m,6'-CH₂ on Gal×3), 4.35-4.46 (2H, m, α-CH on Glu×2), 4.54-4.60 (3H, m,1'-CH on Gal×3), 5.02-5.08 (3H, m, 3'-CH on Gal×3), 5.16-5.22 (3H, m,2'-CH on Gal×3), 5.39 (3H, d, J=3.5 Hz, 4'-CH on Gal×3), 6.93 (1H, d,J=7.5 Hz, NH--CH), 6.99 (1H, t, J=5.5 Hz, NH--CH₂), 7.08 (1H, t, J=5.5Hz, NH--CH₂), 7.22 (1H, d, J=7.5 Hz, NH--CH), 7.68 (1H, t, J=5.5 Hz,NH--CH); IR(nujol): νcm⁻¹ =3280(OH of CONH), 1750 (CH₃ CO), 1635(CONH);FAB-MS:m⁺ /z=1746(M+H⁺)

Example 8

Synthesis of N-(10-hydroxydecanoyl)-L-glutamyl-L-glutamic acidα',α,γ-tri-2-(2',3',4',6'-tetraacetyl-β-D-galactosamine-1)octylamide

(1) Synthesis of 1-(8-azidooctyl)-2,3,4,6-tetraacetyl-β-D-galactosamine

1.35 g of anhydrous ferrous chloride and anhydrous magnesium sulphatewere mixed in 40 ml of methylene chloride, and 1.71 g (10 mmol) of8-azido-octanol and 2.0 g (5.14 mmol) of2-deoxy-2-acetamide-β-D-galactopyranose-tetra-O-acetate were added tothis mixture at room temperature with stirring. After reaction bystirring at room temperature for 6 hours, the admixture was filtered,and the filtrate was washed consecutively with an aqueous saturatedsodium bicarbonate solution and an aqueous saturated sodium chloridesolution. After drying on magnesium sulphate and concentrating undervacuum, the resulting residue was purified by chromatography on silicagel to obtain 1.77 g of the title compound as a colorless caramel-likesolid.

Yield: 68.8% [α]_(D) =-15.9 (C=1.12, 26° C., methanol) NMR (500 MHz, inCDCl₃): δ^(TMS) =1.25-1.40 (8H, brm, --(CH₂)₄ --), 1.55-1.64 (4H, m,--CH₂ --×2), 1.96 (3H, s, CH₃ on Ac), 2.01 (3H, s, CH₃ on Ac), 2.05 (3H,s, CH₃ on Ac), 2.14 (3H, s, CH₃ on Ac), 3.26 (2H, t, J=7 Hz, N₃ -CH₂),3.48 (1H, ddd, J=9.5, 7, and 7 Hz, OCH₂ ×1/2), 3.85-3.95 (3H, m, OCH₂×1/2, 2'-CH on Gal NAc, 5'-CH on GalNAc), 4.13 (1H, dd, J=11 and 7 Hz,6'-CH₂ on GalNAc×1/2), 4.17 (1H, dd, J=11 and 7 Hz, 6'-CH₂ onGalNAc×1/2), 4.72 (1H, d, J=8.5, 1'-CH on GalNAc), 5.28-5.40 (3H, m,3'-CH on GalNAc, 4'-CH on GalNAc); IR(nujol): νcm⁻¹ =3290(NH), 2100(N₃),1750(CH₃ CO); FAB-MS:m/z=501(M+H⁺)

(2) Synthesis of N-(10-hydroxydecanoyl)-L-glutamyl-L-glutamic acidα',α,γ-tri-2-(2',3',4',6'-tetraacetyl-β-D-galactosamine-1)octylamide

1.75 g (3.5 mmol) of the compound obtained in (1) above were dissolvedin 40 ml of ethanol, and 650 mg of methanesulfonic acid and 2 g ofLindlar catalyst were added. The admixture was stirred under a hydrogengas atmosphere under pressure (50 psi) for 2 hours. After adding another1 g of Lindlar catalyst, the admixture was again stirred under ahydrogen gas atmosphere under pressure (50 psi) for 1 hour. The catalystwas removed by filtration, the filtrate was concentrated under vacuum,and the resulting residue was dissolved in 50 ml of dry acetonitrile toobtain an amine solution.

717 mg (1 mmol) of the compound of Synthesis Example 2 were dissolved ina dioxane-water mixture (20 ml-10 ml), and 300 mg of 10%palladium-carbon catalyst were added. The admixture was stirred under ahydrogen gas atmosphere under normal pressure for 20 hours. The catalystwas removed by filtration, the filtrate was concentrated under vacuum,and the resulting residue was dissolved in a dry acetonitrile-DMFmixture (20 ml-7 ml). 1.15 g (10 mmol) of N-hydroxysuccinimide wereadded, and the admixture was stirred while cooled in an ice-ethanolbath. 825 mg of DCC was added, and the reaction was carried out at 0-5°C. for 18 hours. The precipitate was removed by filtration and washedwith a small amount of acetonitrile to obtain an active ester solution.

The amine solution (3.5 mmol) and the active ester solution (1 mmol)were mixed while cooled on an ice bath, 10 mmol of diisopropylethylaminewas added, and the reaction was carried out at 4° C. overnight. Afterconcentration under vacuum, the resulting residue was dissolved in 150ml of ethyl acetate, and washed consecutively with 5% citric acid (15ml×4), an aqueous saturated sodium bicarbonate solution (15 ml×6) and anaqueous saturated sodium chloride solution (15 ml×2). After drying onmagnesium sulphate, the residue was concentrated under vacuum andpurified by chromatography on silica gel (150 g, eluting withchloroform:ethanol=30:1) to obtain 547 mg of the title compound as anamorphous white powder.

Yield: 30.1%. [α]_(D) =-17.6(C=0.99, 27° C., methanol); NMR(500 MHz, inCDCl₃): δ^(TMS) =1.20-1.38 (34H, brm, --(CH₂)₅ -- on --(CH₂)₉ -- and--(CH₂)₄ -- on --(CH₂)8--×3), 1.40-1.66 (16H, brm, --(CH₂)--×8),1.80-2.45 (46H, m, CH₂ CO on decanoyl, CH₃ CO×12, β-CH₂ on Glu×2, andγ-CH₂ on Glu×2), 3.10-3.35 (6H, m, NCH₂ ×3), 3.40-3.52 (3H, m, O--CH₂×1/2×3), 3.60-3.69 (2H, m, changed with D₂ O, HOCH₂), 3.85-4.25 (15H, m,2'-CH on GalNAc×3, 5'-CH on GalNAc×3, 6'-CH₂ on GalNAc×3, and O--CH₂×1/2×3), 4.44-4.55 (2H, brm, NH×2), 4.60-4.73 (3H, m, 1'-CH onGalNAc×3), 5.25-5.42 (6H, m, 3'-CH on GalNAc×3, 4'-CH on GalNAc×3),6.26-6.48 (2H, m, NH×2), 6.83 (1H, d, J=7 Hz, NH), 6.97 (1H, t, J=5 Hz,OH), 7.32-7.45 (2H, m, NH×2), 7.89-7.94 (1H, m, NH); IR(nujol): νcm⁻¹=3280(OH and CONH), 1750(CH₃ CO); FAB-MS:m⁺ /z=1815(M+H⁺)

Example 9

Synthesis of N-(4-hydroxybutyroyl)-L-glutamic acidα,γ-di-{2-(2',3',4',6'-tetraacetyl-β-D-galactosyl-1)ethoxy}ethoxyethylamide

(1) Synthesis of N-(4-benzyloxybutyroyl)-L-glutamic acid α,γ-dibenzylester

7.97 g (16 mmol) of L-glutamic acid α,γ-dibenzyl esterp-toluenesulfonate were dissolved in 30 ml of dry acetonitrile, and 4 mlof diisopropylethylamine was added while cooled in an ice water bath toobtain an amine solution.

1.5 g of 4-benzyloxybutyric acid were dissolved in dry acetonitrile-DMF(30 ml-9 ml). 2.13 g of N-hydroxysuccinimide were added, and theadmixture was stirred while cooled in an ice water bath. 1.91 g of DCCwere added, and the reaction was carried out for 5 hours. Theprecipitate was removed by filtration and washed with a small amount ofacetonitrile to obtain an active ester solution.

The amine solution and the active ester solution thus prepared weremixed while cooled in an ice bath, and the reaction was carried out at0° C. for 2 hours. After concentration under vacuum, the resultingresidue was dissolved in chloroform, and washed consecutively with a 5%aqueous citric acid solution, an aqueous saturated sodium bicarbonatesolution, an aqueous saturated sodium hydrogen carbonate solution and anaqueous saturated sodium chloride solution. After drying on magnesiumsulphate anhydrous, the residue was concentrated under vacuum andpurified by chromatography on silica gel (200 g, eluting with methylenechloride:ethanol=50:1-20:1) to obtain 1.59 g of the title compound as anamorphous yellow powder.

Yield: 41%. [α]_(D) ²⁴ -16.4 (c0.88, MeOH); IR (CHCl₃): 1735cm⁻¹,1674cm⁻¹, 1171cm⁻¹ ; ¹ H-NMR(CDCl₃)δ: 7.35-7.31 (15H, m, Ph-H×3), 6.31(1H, brs, NH), 5.15 (2H, slike, CH₂ Ph), 5.10,5.07 (each 1H, J=14.5 Hz,CH₂ Ph), 4.67-4.63 (1H, m, Glu-α), 4.49,4.46 (each 1H, J=12.0 Hz, CH₂Ph), 3.50 (1H, tlike, Glu-γ), 2.44-2.16 (5H, m, etylene moiety),1.99-1.89 (3H, m, etylene moiety, Glu-β)

(2) Synthesis of N-(4-hydroxybutyroyl)-L-glutamic acidα,γ-di-{(2--(2',3',4',6'-tetraacetyl-β-D-galactosyl-1)ethoxy}ethoxyethylamide

2.8 g (5.6 mmol) of1-(2'-azidoethoxyethoxyethyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranosewere dissolved in 60 ml of ethanol, and 761 mg (8.4 mmol) ofmethanesulfonic acid and 3 g of Lindlar catalyst were added. Theadmixture was stirred under a hydrogen gas flow under pressure (50 psi)for 2 hours. After adding another 1.5 g of Lindlar catalyst, theadmixture was again stirred under a hydrogen gas flow under pressure (50psi) for 1 hour. The catalyst was removed by filtration, the filtratewas concentrated under vacuum, and the resulting residue was dissolvedin 50 ml of dry acetonitrile to obtain an amine solution.

1.0 g (2.0 mmol) of the compound of (1) above was dissolved in adioxane-water mixture (20 ml-7.5 ml), and 300 mg of 10% palladium-carboncatalyst were added. The admixture was stirred under a hydrogen gas flowunder normal pressure for 12 hours. The catalyst was removed byfiltration, the filtrate was concentrated under vacuum, and theresulting residue was dissolved in a dry acetonitrile-DMF mixture (25ml-7.5 ml). 1.1 g (9.6 mmol) of N-hydroxysuccinimide were added, and theadmixture was stirred while cooled in an ice-water bath. 1.0 g (4.8mmol) of DCC was added, and the reaction was carried out for 4 hours.The precipitate was removed by filtration and washed with a small amountof acetonitrile to obtain an active ester solution.

The abovementioned amine solution (5.6 mmol) and the active estersolution (2.0 mmol) were mixed while cooled on an ice-water bath, 2.5 mlof diisopropylethylamine was added, and the reaction was carried out at0° C. for 14 hours. After concentration under vacuum, the resultingresidue was dissolved in chloroform, and washed consecutively with a 5%aqueous citric acid solution, an aqueous saturated sodium chloridesolution, an aqueous saturated sodium bicarbonate solution and anaqueous saturated sodium chloride solution. After drying on anhydrousmagnesium sulphate, the residue was concentrated under vacuum andpurified by chromatography on silica gel (60 g, eluting withchloroform:ethanol=15:1) to obtain 707 mg of the title compound as anamorphous orange powder. [α]_(D) ²⁴⁻ 10.7° (c1.22, MeOH) IR(CHCl₃): 1749cm⁻¹, 1661 cm⁻¹, 1078 cm⁻¹ ; ¹ H-NMR(CDCl₃) δ: 7.43 (1H, t, J=5.6 Hz,NHCO), 7.31 (1H, d, J=9.3 Hz, NHCO), 6.62 (1H, t, J=5.4 Hz, NHCO), 5.40(2H, d, J=3.4 Hz, Gal-4×2), 5.22-5.18 (2H, m, Gal-2×2), 5.05-5.02 (2H,m, Gal-3×2), 4.57-4.55 (2H, m, Gal-1×2), 4.42-4.37 (1H, m, Glu-α),4.21-4.11 (4H, m, Gal-6×2), 4.00-3.96 (2H, m, etyleneglycol moiety),3.95-3.92 (2H, m, Gal-5×2), 3.76-3.35 (22H, m, ethylenglycol moiety),3.24 (1H, brs, OH), 2.43-2.27 (6H, m, Glu-g, ethylene moiety, Glu-b),2.16, 2.06, 2.05, 1.99 (each s, 6H, acetyl), 1.97-1.82 (2H, m, etylenemoiety)

Reference Example 1

Synthesis ofN-(4-hydroxybutyroyl)-{2'-(2',3',4',6'-tetraacetyl-β-D-galactosyl-1)-ethoxy}ethoxyethylamide

(1) Synthesis ofN-(4-benzyloxybutyroyl)-{2'-(2',3',4',6'-tetraacetyl-β-D-galactosyl-1)ethoxy}ethoxyethylamide

1.0 g (2.0 mmol) of1-(2'-azidoethoxyethoxyethyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranosewas dissolved in 20 ml of ethanol, and 285 mg of methanesulfonic acidand 1.0 g of Lindlar catalyst were added. The admixture was stirredunder a hydrogen gas atmosphere under pressure (50 psi) for 2 hours.After adding another 0.5 g of Lindlar catalyst, the admixture was againstirred under a hydrogen gas atmosphere under pressure (50 psi) for 2hour.

The catalyst was removed by filtration, the filtrate was concentratedunder vacuum, and the resulting residue was dissolved in dryacetonitrile to obtain an amine solution.

256 mg (1.3 mmol) of 4-benzyloxybutyric acid were dissolved in a dryacetonitrile-DMF mixture (5 ml-1.5 ml). 365 mg (3.2 mmol) ofN-hydroxysuccinimide were added, and the admixture was stirred whilecooled in an ice-water bath. 326 mg (1.6 mmol) of DCC were added, andthe reaction was carried out at 0° C. for 5 hours. The precipitate wasremoved by filtration and washed with a small amount of acetonitrile toobtain an active ester solution.

The amine solution (2.0 mmol) and the active ester solution (1.3 mmol)were mixed while cooled on an ice-water bath, 1 ml ofdiisopropylethylamine was added, and the reaction was carried out at 0°C. overnight. After concentration under vacuum, the resulting residuewas dissolved in chloroform, and washed consecutively with a 5% aqueouscitric acid solution, an aqueous saturated sodium bicarbonate solutionand an aqueous saturated sodium chloride solution. After drying onanhydrous magnesium sulphate, the residue was concentrated under vacuumand purified by chromatography on silica gel (5.0 g, eluting withmethylene chloride:methanol=20:1) to obtain 429 mg of the title compoundas a pale yellow oil.

Yield: 30%. [α]_(D) ²³ -9.1° (c1.00, CHCl₃); IR(CHCl₃): 1749 cm⁻¹, 1664cm⁻¹, 1078cm⁻¹ ; ¹ H-NMR(CDCl₃) δ:

7.36-7.27 (5H, m, Ph-H), 6.12 (1H, brs, NH), 5.39 (1H, dlike, Gal-4),5.21 (1H, dd, J=7.9 Hz, J=10.5 Hz, Gal-2), 5.02 (1H, dd, J=2.7 Hz,J=10.5 Hz, Gal-3), 4.53 (1H, d, J=7.9 Hz, Gal-1), 4.50 (2H, slike, CH₂Ph), 4.17 (1H, dd, J=11.5 Hz, J=6.6 Hz, Gal-6), 4.12 (1H, dd, J=11.5 Hz,J=6.6 Hz, Gal-6), 3.99-3.95 (1H, m), 3.89 (1H, tlike, Gal-5), 3.74-3.70(1H, m), 3.65-3.51 (10H, m), 3.45-3.42 (2H, m, CH₂ CONH), 2.31 (2H,tlike, CH₂ NH), 2.14, 2.05, 2.05, 1.99 (each 3H, s, acetyl), 1.97-1.94(2H, m, CH₂ OCH₂ Ph)

(2) Synthesis ofN-(4-hydroxybutynoyl)-{2'-(2',3',4,6'-tetraacetyl-β-D-galactosyl-1)ethoxy}ethoxyethylamide

200 mg (2.4 mmol) of the compound of (1) above were dissolved in 5 ml ofethyl acetate, and 10% palladiumcarbon was added. The admixture wasstirred under a hydrogen gas atmosphere under normal pressure for 3hours. The catalyst was removed by filtration, the filtrate wasconcentrated under vacuum, and the resulting residue was purified bychromatography on silica gel (2 g, methylene chloride:methanol=20:1) toobtain 117 mg of the title compound as a colorless oil.

Yield: 86% [α]_(D) ²⁴ -5.9° (c1.00, MeOH); IR(CHCl₃): 3450 cm⁻¹, 1749cm⁻¹, 1651 cm⁻¹, 1078 cm⁻¹ ; ¹ H-NMR(CDCl₃)δ: 6.49 (1H, brs, NH), 5.39(1H, dlike, Gal-4), 5.20 (1H, dd, J=8.0 Hz, J=10.5 Hz, Gal-2), 5.03 (1H,dd, J=3.4 Hz, J=10.5 Hz, Gal-3), 4.55 (1H, d, J=8.0 Hz, Gal-1), 4.19(1H, dd, J=6.5 Hz, J=11.2 Hz, Gal-6), 4.12 (1H, dd, J=6.8 Hz, J=11.2 Hz,Gal-6), 4.02-3.98 (1H, m, ethyleneglycol moiety), 3.92 (1H, tlike,Gal-5), 3.76-3.59 (9H, m, etyleneglycol moiety), 3.56 (2H, tlike, CH₂NH), 3.48-3.44 (2H, m, CH₂ OH), 3.20-3.02 (1H, brs, OH), 2.39 (2H,tlike, CH₂ CONH), 2.16, 2.07, 2.05, 1.99 (each 3H, s, acetyl), 1.91-1.86(2H, m, CH₂ CH₂ OH)

Example 10

Synthesis of Phosphoroamidite (1)

175 mg (0.1 mmol) of the compound of Example 4 were dissolved in 20 mlof dry methylene chloride, and 100 mg (0.33 mmol) of2-cyanoethyl-N,N,N',N'-tetraisopropyl-phosphoroamidite were added whilestirring on an ice-ethanol bath under a nitrogen gas flow. 0.3 ml of a0.5 M tetrazole-acetonitrile solution was added dropwise at -5 to -10°C. After stirring at room temperature for 2.5 hours, 30 ml of a cooled 1M aqueous triethyl ammonium hydrogencarbonate solution were added, andthe admixture was further stirred for 10 minutes. The organic layer wasisolated, washed consecutively with a 1 M aqueous triethyl ammoniumhydrogencarbonate solution (10 ml) and an aqueous saturated sodiumchloride solution, dried on magnesium sulphate, and concentrated undervacuum. The resulting residue was washed with hexane (30 ml×3) and driedunder vacuum to obtain 226 mg of phosphoroamidite as an amorphous whitepowder.

Yield: quantitative. NMR(500 MHz, in CDCl₃): δ^(TMS) =1.18-1.40 (34H, m,-CH₃ on iPrN×4, --(CH₂)₅ -- on --(CH₂)₉ -- and --(CH₂)₂ -- on --(CH₂)₆--×3), 1.44-1.66 (16H, m, --(CH₂)--×8), 1.85-2.57 (44H, m, CH₃ CO×12 andβ-CH₂ on Glu×2, γ-CH₂ on Glu×2 and CH₂ CO on decanoyl), 2.65 (2H, t,J=6.5 Hz, CH₂ CN), 3.10-3.36 (6H, m, NCH₂ ×3), 3.42-3.70 (9H, m, P-O-CH₂×2, NCH on iPr×2, and OCH₂ -Gal=1/2×3), 3.75-3.94 (6H, m, 5'-CH onGal×3,and OCH₂ -Gal×1/2×3), 4.08-4.24 (6H, m, 6'-CH₂ on Gal×3), 4.38(1H, m, α-CH on Glu), 4.46 (3H, d, J=8 Hz, 1'-CH on Gal×3), 4.64 (1H, m,α-CH on Glu), 5.00-5.05 (3H, m, 3'-CH on Gal×3), 5.16-5.22 (3H, m, 2'-CHon Gal×3), 5.39 (3H, m, 4'-CH on Gal×3), 6.24 (1H, t, J=5 Hz, NH--CH₂),6.35 (1H, t, J=7 Hz, NH--CH), 6.48 (1H, t, J=5 Hz, NH--CH₂), 6.68 (1H,d, J=7 Hz, NH--CH), 7.75 (1H, J=5 Hz, NH--CH₂)

Example 11

Synthesis of Phosphoroamidite (2)

783 mg (0.5 mmol) of the compound of Example 5 were dissolved in 20 mlof dry methylene chloride, and 226 mg (0.75 mmol) of2-cyanoethyl-N,N,N',N'-tetraisopropyl-phosphoroamidite were added whilestirring on an ice-ethanol bath under a nitrogen gas flow. 1 ml of a 0.5M tetrazole-acetonitrile solution was added dropwise at -5 to -10° C.After stirring at room temperature for 1 hour, 50 ml of a cooled 1 Maqueous triethyl ammonium hydrogencarbonate solution were added, and theadmixture was further stirred for 10 minutes. The organic layer wasisolated, washed consecutively with a 1 M aqueous triethyl ammoniumhydrogencarbonate solution (10 ml) and an aqueous saturated sodiumchloride solution, dried on magnesium sulphate, and concentrated undervacuum. The resulting residue was washed with hexane (30 ml×3) and driedunder vacuum to obtain 833 mg of phosphoroamidite as an amorphous whitepowder.

Yield: 94.3%. NMR(500 MHz, in CDCl₃): δ^(TMS) =1.17 (6H, d, J=6 Hz,--(CH₃)₂ on iPrN), 1.18 (6H, d, J=6 Hz, --(CH₃)₂ on iPrN), 1.20-1.38(10H brm, --(CH₂)₅ --), 1.55-1.66 (4H, m, --(CH₂)₂ --), 1.90-2.18 (42H,m, CH₃ CO×12 and β-CH₂ on Glu×2 and CH₂ CO on decanoyl), 2.28-2.44 (4H,m, γ-CH₂ on Glu×2), 2.65 (2H, t, J=6.5 Hz, CH₂ CN), 3.35-3.53 (6H, m,NCH₂ ×3), 3.53-3.74 (7H, m, P--O--CH₂, NCH on iPr×2, and OCH₂-Gal×1/2×3), 3.75-3.98 (8H, m, 5'-CH on Gal×3, CH₂ CH₂ CN and OCH₂-Gal×1/2×3), 4.07-4.22 (6H, m, 6'-CH₂ on Gal×3), 4.40-4.52 (2H, m, α-CHon Glu×2), 4.50-4.55 (3H, m, 1'-CH on Gal×3), 5.00-5.06 (3H, m, 3'-CH onGal×3), 5.13-5.19 (3H, m, 2'-CH on Gal×3), 5.35-5.45 (3H, m, 4'-CH onGal×3), 6.56 (1H, d, J=7.5 Hz, NH--CH), 6.88 (1H, t, J=6 Hz, NH--CH₂),6.95 (1H, t, J=5.5 Hz, NH--CH₂), 7.13 (1H, d, J=7.5 Hz, NH--CH), 7.72(1H, J=5.5 Hz, NH--CH₂); IR(nujol): νcm⁻¹ =3290(CONH), 1755(CH₃ CO),1635(CONH)

Example 12

Synthesis of Phosphoroamidite (3)

641 mg (0.4 mmol) of the compound of Example 7 were dissolved in 20 mlof dry methylene chloride, and 181 mg (0.6 mmol) of2-cyanoethyl-N,N,N',N'-tetraisopropyl-phosphoroamidite were added whilestirring on an ice-ethanol bath under a nitrogen gas flow. 1 ml of a 0.5M tetrazole-acetonitrile solution was added dropwise at -5 to -10° C.After stirring at room temperature for 3 hours, 40 ml of a cooled 1 Maqueous triethyl ammonium hydrogencarbonate solution were added, and theadmixture was further stirred for 10 minutes. The organic layer wasisolated, washed consecutively with a 1 M aqueous triethyl ammoniumhydrogencarbonate solution (10 ml) and an aqueous saturated sodiumchloride solution, dried on magnesium sulphate, and concentrated undervacuum. The resulting residue was washed with hexane (30 ml×3) and driedunder vacuum to obtain 752 mg of phosphoroamidite as an amorphous whitepowder.

Yield: quantitative. NMR (500 MHz, in CDCl₃): δ^(TMS) =1.17 (6H, d,J=6.5 Hz, --(CH₃)₂ on iPrN), 1.18 (6H, d, J=6.5 Hz, --(CH₃)₂ on iPrN),1.88-2.20 (42H, m, CH₃ CO×12 and β-CH₂ on Glu×2 and CH₂ --CH₂ --O--P),2.26-2.43 (6H, m, γ-CH₂ on Glu×2 and CH₂ CO on butyloyl), 2.66 (2H, t,J=6.5 Hz, CH₂ CN), 3.30-3.92 (39H, m, NCH₂ ×3, OCH₂ ×12, NCH on iPr×2,OCH₂ on butyloyl, CH₂ CH₂ CN, and OCH₂ -Gal×1/2×3), 3.90-4.00 (6H, m,5'-CH on Gal×3 and OCH₂ -Gal×1/2×3), 4.09-4.24 (6H, m, 6'-CH₂ on Gal×3),4.33-4.47 (2H, m, α-CH on Glu×2), 4.54-4.60 (3H, m, 1'-CH on Gal×3),5.02-5.08 (3H, m, 3'-CH on Gal×3), 5.16-5.24 (3H, m, 2'-CH on Gal×3),5.39 (3H, d, J=3.5 Hz, 4'-CH on Gal×3), 6.60 (1H, d, J=7.5 Hz, NH--CH),7.02-7.07 (1H, m, NH--CH₂), 7.10-7.16 (1H, m, NH--CH₂), 7.23 (1H, d,J=7.5 Hz, NH--CH), 7.80-7.86 (1H, m, NH--CH)

Example 13

Synthesis of Phosphoroamidite (4)

181 mg (0.1 mmol) of the compound of Example 8 were dissolved in 20 mlof dry methylene chloride, and 100 mg (0.33 mmol) of2-cyanoethyl-N,N,N',N'-tetraisopropyl-phosphoroamidite were added whilestirring on an ice-ethanol bath under a nitrogen gas flow. 0.3 ml of a0.5 M tetrazole-acetonitrile solution was added dropwise at -5 to -10°C. After stirring at room temperature for 2.5 hours, 30 ml of a cooled 1M aqueous triethyl ammonium hydrogencarbonate solution were added, andthe admixture was further stirred for 10 minutes. The organic layer wasisolated, washed consecutively with a 1 M aqueous triethyl ammoniumhydrogencarbonate solution (10 ml) and an aqueous saturated sodiumchloride solution, dried on magnesium sulphate, and concentrated undervacuum. The resulting residue was washed with hexane (30 ml×3) and driedunder vacuum to obtain 168 mg of phosphoroamidite as an amorphous whitepowder.

Yield: 85%. NMR (500 MHz, in CDCl₃): δ^(TMS) =1.18-1.38 (46H, m, --CH₃on iPrN×4, --(CH₂)₅ -- on --(CH₂)₉ -- and --(CH₂)₄ -- on --(CH₂)₆ --×3),1.44-1.70 (16H, m, --(CH₂)--×8), 1.85-2.45 (44H, m, CH₃ CO×12 and β-CH₂on Glu×2, γ-CH₂ on Glu×2 and CH₂ CO on decanoyl), 2.65 (2H, t, J=6.5 Hz,CH₂ CN), 3.10-3.36 (6H, m, NCH₂ ×3), 3.40-3.50 (7H, m, P--O--CH₂, NCH oniPr×2, and OCH₂ -Gal×1/2×3), 3.76-4.24 (17H, m, 2'-CH on GalNAc×3, 5'-CHon GalNAc×3, 6'-CH₂ on GalNAc×3, O-CH₂ --CH₂ CN, and OCH₂ -Gal×1/2×3),4.40-4.60 (2H, m, α-CH on Glu×2), 4.58-4.74 (3H, m, 1'-CH on GalNAc×3),5.25-5.40 (6H, m, 3'-CH on Gal×3, and 4'-CH on Gal×3), 6.26-6.40 (2H, m,NH×2), 6.80 (1H, d, J=7 Hz, NH--CH), 7.39 (1H, d, J=7 Hz, NH--CH), 7.94(1H, m, NH)

Example 14

Synthesis of Phosphoroamidite (5)

116 mg (0.10 mmol) of the compound of Example 9 were dissolved in 5 mlof dry methylene chloride, and 48 mg (0.16 mmol) of2-cyanoethyl-N,N,N',N'-tetraisopropyl-phosphoroamidite were added whilestirring on an ice-ethanol bath under an argon gas flow. 0.28 ml of a0.5 M 1H-tetrazole-acetonitrile solution was added dropwise at -5 to-10° C. After stirring at room temperature for 3 hours, 5 ml of a cooled1 M aqueous triethyl ammonium hydrogencarbonate solution were added, andthe admixture was further stirred for 10 minutes. The organic layer wasisolated, washed consecutively with a 1 M aqueous triethyl ammoniumhydrogencarbonate solution and an aqueous saturated sodium chloridesolution, dried on anhydrous magnesium sulphate, and concentrated undervacuum. The resulting residue was washed with dry hexane and dried undervacuum to obtain 232 mg of phosphoroamidite as a white viscoussubstance.

Reference Example 2

Synthesis of Phosphoroamidite (6)

73 mg (0.13 mmol) of the compound of Reference Example 1 were dissolvedin 5 ml of dry methylene chloride, and 63 mg (0.21 mmol) of2-cyanoethyl-N,N,N',N'-tetraisopropyl-phosphoroamidite were added whilestirring on an ice-ethanol bath under an argon gas flow. 0.36 ml of a0.5 M 1H-tetrazole-acetonitrile solution was added dropwise at -5 to-10° C. After stirring at room temperature for 3 hours, 5 ml of a cooled1 M aqueous triethyl ammonium hydrogencarbonate solution were added, andthe admixture was further stirred for 10 minutes. The organic layer wasisolated, washed consecutively with a 1 M aqueous triethyl ammoniumhydrogencarbonate solution and an aqueous saturated sodium chloridesolution, dried on magnesium sulphate, and concentrated under vacuum.The resulting residue was washed with dry hexane and dried under vacuumto obtain 98 mg of phosphoroamidite as a white viscous substance.

¹ H-NMR (CDCl₃) δ: 6.50-6.46 (1H, m, NHCO), 5.39 (1H, brs, Gal-4),5.22-5.18 (1H, m, Gal-2), 5.04-5.00 (1H, m, Gal-3), 4.56 (1H, d, J=7.5Hz, Gal-1), 4.32-4.31 (1H, m, Gal-OCH₂ ×1/2), 4.21-4.11 (2H, m, Gal-6),3.99-3.87 (2H, m, Gal-5, Gal-OCH₂ ×1/2), 3.75-3.55 (12H, m,etyleneglycol moiety, NCH(CH₃)₂ ×2, CH₂ CH₂ CN), 2.67-2.66 (2H, m, CH₂CN), 2.35-2.30 (2H, m, CH₂ CONH), 2.16, 2.06, 2.05, 1.99 (each 3H, s,acetyl), 1.97-1.95 (2H, m, CH₂ CH₂ CONH, CH₂ OP), 1.50-1.49 (12H, dlike,CH(CH₃)₂ ×2)

Reference Example 3

Synthesis of Phosphoroamidite (7)

1-β-(2'-hydroxyethyl)-2,3,4,6-tetra-O-acetyl-galactose (280 mg, 0.71mmol) was dissolved in dry dichloromethane (10 ml), a 0.5 Mtetrazole/acetonitrile solution (1.4 ml) was added under an argonatmosphere, a phosphorylation reagent (350 μl, 1.1 mmol) was added, andthe admixture was stirred at room temperature for 2 hours. The reactionsolution was poured into a 0.5 M aqueous TEAB solution (50 ml) andextracted with dichloromethane. The organic layer was dried on magnesiumsulphate, and the solvents were removed by distillation to obtain thephosphoroamidite.

Yield: quantitative. ¹ H-NMR(CDCl₃) δ: 5.39 (1H, d, J₃.4 =3.5 Hz, H-4),5.20 (1H, ddd, J₃.4 =4.0 Hz, J₂.3 =8.0 Hz, J=11.0 Hz, H-2), 5.02 (1H,ddd, J₃.4 =3.5 Hz, J₂.3 =7.0 Hz, J=14.0 Hz, H-3), 4.59 (1H, dd, J₁.2=8.0 Hz, J=11.0 Hz, H-1), 4.10-4.20 (3H, m), 3.70-4.00 (6H, m),3.59-3.62 (2H, m), 2.63-2.67 (2H, m), 2.15 (3H, s, OAc), 2.05 (3H, s,OAc), 2.05 (3H, s, OAc), 1.99 (3H, s, OAc), 1.17-1.20 (12H, m)

Structural details of each of the compounds of Examples 1 to 14 inrelation to formula (I) are shown in Table 2.

                                      TABLE 2                                     __________________________________________________________________________    Compounds of Example 1-14                                                     Example                                                                            X    m (T.sup.5)r                                                                          n p T.sup.3                                                                             T.sup.1                                                                          F.sup.1                                                                            q T.sup.4                                                                             T.sup.2                                                                            F.sup.2                      __________________________________________________________________________    1    benzyloxy                                                                          0 --CONH--                                                                            0 0 --CONH--                                                                            t = 2                                                                            Gal  2 --CONH--                                                                            t = 2                                                                              Gal                            2 t-butoxy 0 --CONH-- 0 0 --CONH-- t = 2 Gal 2 --CONH-- group(IV) Gal                                                         3 t-butoxy 0 --CONH-- 0                                                      0 --CONH-- t = 2 Glc 2                                                        --CONH-- group(IV) Glc                                                         4 HC 9 --CONH-- 0 0                                                          --CONH-- s = 6 Gal 2                                                          --CCNH-- group(IV) Gal                                                         5 HO 9 --CONH-- 0 0                                                          --CONH-- s = 2 Gal 2                                                          --CONH-- group(IV) Gal                                                         6 HO 3 --CONH-- 0 0                                                          --CONH-- s = 2 Gal 2                                                          --CONH-- group(IV) Gal                                                         7 HQ 3 --CONH-- 0 0                                                          --CONH-- t = 2 Gal 2                                                          --CONH-- group(IV) Gal                                                         8 HO 9 --CONH-- 0 0                                                          --CONH-- s = 8 GalNAC 2                                                       --CONH-- group(IV)                                                            GalNAC                         9 HO 3 --CONH-- 0 0 --CONH-- t = 2 Gal 2 --CONH-- t = 2 Gal                   10 group(II) 9 --CONH-- 0 0 --CONH-- s = 6 Gal 2 --CONH-- group(IV) Gal       11 group(II) 9 --CONH-- 0 0 --CONH-- s = 2 Gal 2 --CONH-- group(IV) Gal       12 group(II) 3 --CONH-- 0 0 --CONH-- t = 2 Gal 2 --CONH-- group(IV) Gal       13 group(II) 9 --CONH-- 0 0 --CONH-- s = 8 GalNAC 2 --CONH-- group(IV)                                                       GalNAc                         14 group(II) 3 --CONH-- 0 0 --CONH-- t = 2 Gal 2 --CONH-- t = 2             __________________________________________________________________________                                                     Gal                      

In Table 2, Gal represents galactose, Glc represents glucose and GalNAcrepresents N-acetyl-galactosamine (same in Table 3 hereinafter). Y ingroup (II) is diisopropyl amino group.

In compounds in which T² is group (IV), structures of group (VI) are asfollows:

                  TABLE 3                                                         ______________________________________                                        Group (IV)                                                                      Exam-                                                                         ple p* T.sup.3 * T.sup.1 * q* T.sup.4 * T.sup.1 ** F.sup.3                  ______________________________________                                        2     0     --CONH--  t = 2                                                                              2   --CONH--                                                                              t = 2                                                                              Gal                                 3 0 --CONH-- t = 2 2 --CONH-- t = 2 Glc                                       4 0 --CONH-- s = 6 2 --CONH-- s = 6 Gal                                       5 0 --CONH-- s = 2 2 --CONH-- s = 2 Gal                                       6 0 --CONH-- s = 2 2 --CONH-- s = 2 Gal                                       7 0 --CONH-- t = 2 2 --CONH-- t = 2 Gal                                       8 0 --CONH-- s = 8 2 --CONH-- s = 8 GalNAc                                    10 0 --CONH-- s = 6 2 --CONH-- s = 6 Gal                                      11 0 --CONH-- s = 2 2 --CONH-- s = 2 Gal                                      12 0 --CONH-- t = 2 2 --CONH-- t = 2 Gal                                      13 0 --CONH-- s = 8 2 --CONH-- s = 8 GalNAc                                 ______________________________________                                    

Example 15

Synthesis of Nucleotide Derivative (1) (1) Synthesis of TetrathymidineNucleotide

Using an automated DNA synthesizer (Cyclone Plus Nucleic AcidSynthesizer, a product of Milipore), tetrathymidine nucleotide wassynthesized on a synthesizing column (15 μmol synthesis scale) accordingto the β-cyanoethylphosphoroamidite method. Peaction programs andsynthesizing reagents provided by or purchased from Milipore were usedwithout alteration.

After completion of the reaction, the column was washed with 10 ml ofpurified water, the carrier was removed from the column, 5 ml ofconcentrated aqueous ammonia (25%) was added, and the admixture wasallowed to stand at room temperature for 24 hours. The carrier wasremoved by decantation, the supernatant was concentrated under vacuum,and the resulting supernatant was concentrated under vacuum, 0.7 ml of100 mM TEAA (pH 8.0) was added to the residue. After filtration,purification was carried out using HPLC under the following conditions:

[Conditions for HPLC]

Column: ODS-packed column (ODS-2 column, 250×6 mm, a product of G.L.Science)

Column temperature: 30° C.

Detection: OD (260 nm)

Flow rate: 1.5 ml/min

Sample volume: 250 μl

Moving phase A: 100 mM TEAA (pH 6.1)

Moving phase B: 95% acetonitrile

0 minute: A:B=95:5; 40 minutes: A:B=80:20; linear gradient

Moving phases which were eluted in a designated retention time werepooled.

Retention time: 25.0-26.0 minutes (1 minute)

A pooled fraction of moving phases was dried under vacuum. The residuewas dissolved in 1 ml of 100 mM TEAA, and the solution was added to anODS column (Sep-Pak Plus, a product of Milipore) equilibrated with 100mM TEAA containing 10% acetonitrile.

The column was washed with the solution used for equilibration (5 ml×3)and then with purified water (5 ml×3), and eluted with 70% acetonitrile.The eluate was dried under vacuum to obtain a white amorphous powder.

Yield: 27.0% NMR (500 MHz, in D₂ O): δ^(TMS) =1.87, 1.88, 1.89 (total12H, each singlet, CH₃ on Thymine), 2.28-2.37 (4H, m, 2'-CH₂ ×4×1/2),2.48-2.56 (4H, m, 2'-CH₂ ×4×1/2), 3.79 (1H, dd, J=4 and 12 Hz,5'-terminal 5'-CH₂ ×1/2), 3.82 (1H, dd, J=4 and 12 Hz, 5'-terminal5'-CH₂ ×1/2), 4.04-4.20 (8H, m), 4.28-4.34 (2H, m), 4'-CH×4 and 5'-CH₂×3, 4.58 (1H, q, J=4 Hz, 3'-terminal 3'-CH), 4.83-4.92 (3H, m, 3'-CH×3),6.20 (1H, dd, J=6.5 and 7 Hz, 1'-CH), 6.23-6.32 (3H, m, 1'-CH×3), 7.64(1H, s, CH on Thymine), 7.66 (1H, s, CH on Tyhmine), 7.68 (1H, s, CH onThymine), 7.69 (1H, s, CH on Thymine); FAB-MS:m⁺ /z=1177(M-H⁺)

(2) Synthesis of Ethoxy-β-galactose-modified Tetrathymidine Nucleotide

The galactose derivative of phosphoroamidite (Reference Example 3) wasdissolved in acetonitrile to a concentration of 60 mM, and the solutionwas immediately applied onto an automated DNA synthesizer. It was thenreacted with tetrathymidine nucleotide previously synthesized on acolumn for synthesis (15 μmol synthesis scale) according to theβ-cyanoethylphosphoroamidite method. After completion of the reaction, awhite amorphous powder was obtained in the same manner as described in(1).

Conditions for HPLC were the same as described in (1). The retentiontime for the pooled fraction was 20.5-21.5 minutes (for 1 minute).

Yield: 39.8% NMR (500 MHz, in D₂ O): δ^(TMS) =1.89 (6H, s, CH₃ onThymine×2), 1.91 (3H, s, CH₃ on Thymine), 1.92 (3H, s, CH₃ on Thymine),2.28-2.40 (4H, m, 2'-CH₂ ×4×1/2), 2.48-2.57 (4H, m, 2'-CH₂ ×4×1/2), 3.54(1H, dd, J=8 and 9 Hz, 2"-CH on Gal), 3.63-3.90 (4H, m, 3"-CH, 5"-CH,6"-CH₂ on Gal), 3.92 (1H, m, 4"-CH on Gal), 4.02-4.20 (10H m), 4.29-4.39(2H, m), 4'-CH×4 and 5'-CH₂ ×4, 4.44 (1H, d, J=8 Hz, 1"-CH on Gal),4.57-4.62 (1H, m, 3'-CH), 4.85-4.94 (3H, m, 3'-CH×3), 6.22-6.35 (4H, m,1'-CH×4), 7.67 (1H, s, CH on Thymine), 7.68 (1H, s, CH on Thymine), 7.71(1H, s, CH on Thymine), 7.72 (1H, s, CH on Thymine)

Example 16

Synthesis of Nucleotide Derivative (2)

Synthesis of tri(ethoxy-β-galactose)-modified Tetrathymidine Nucleotide

The galactose derivative of phosphoroamidite (Example 11) was dissolvedin acetonitrile to a concentration of 60 mM, and the solution wasimmediately applied onto an automated DNA synthesizer. It was thenreacted with tetrathymidine nucleotide previously synthesized on asynthesizing column (15 μmol synthesis scale) according to theβ-cyanoethylphosphoroamidite method. After completion of the reaction, awhite amorphous powder was obtained in the same manner as described inExample 15 (1).

Conditions for HPLC were the same as described in Example 15 (1), exceptthat the moving phase mixing ratios were altered as followed: 0 minute:A:B=95:5; 40 minutes: A:B=70:30; a linear gradient. The retention timefor the pooled fraction was 21.5-22.5 minutes (for 1 minute).

Yield: 17.4% NMR (500 MHz, in D₂ O): δ^(TMS) =1.00-1.25 (10H, m,--(CH₂)₅ -- on decanoyl), 1.48-1.58 (4H, m, --(CH₂)₂ -- on decanoyl),1.90 (3H, d, J=1 Hz, CH₃ on Thymine), 1.91 (3H, d, J=1 Hz, CH₃ onThymine), 1.92 (3H, d, J=1 Hz, CH₃ on Thymine), 1.93 (3H, d, J=1 Hz, CH₃on Thymine), 1.97 (2H, m, βCH₂ on Glu×2), 2.11 (2H, m, βCH₂ on Glu×2),2.28 (2H, t, J=7 Hz, --CH₂ CO on decanoyl), 2.20-2.58 (12H, m, 2'-CH₂ ×4on dRib and γCH₂ on Glu×2), 3.38-3.57 (6H, m, NHCH₂ on ethyleneglycol×3), 3.54 (3H, m, 2"-CH on Gal×3), 3.64-4.02 (18H, m, OCH₂ onethylene glycol×3, and 3"-CH, 5"-CH, 6"-CH₂ on Gal×3), 3.93 (3H, m,4"-CH on Gal×3), 4.06-4.18 and 4.30-4.40 (12H, m, 4'-CH and 5'-CH₂ ondRib×4), 4.24-4.30 (2H, m, αCH on Glu×2), 4.41 (3H, m, 1"-CH on Gal×3),4.57-4.62 and 4.85-4.94 (4H, m, 3'-CH on dRib×4), 6.25-6.35 (4H, m,1'-CH on dRib×4), 7.69 (1H, d, J=1 Hz, CH on Thymine), 7.71 (1H, d, J=1Hz, CH on Thymine), 7.74 (1H, d, J=1 Hz, CH on Thymine), 7.77 (1H, d,J=1 Hz, CH on Thymine)

Example 17

Synthesis of Phosphorothioate Oligodeoxynucleotide Derivative (1)

(1) Synthesis of Tetrathymidine Phosphorothioate Oligodeoxynucleotide

A white amorphous powder was obtained in the same manner as described inExample 15 (1), except that, among synthesizing reagents to be used inthe automated DNA synthesizer, the oxidizing solution was replaced byBeaucage reagent (a product of Milipore) for thio conversion.

Conditions for HPLC were the same as described in Example 15 (1), exceptthat the moving phase mixing ratios were altered as follows: 0 minute:A:B=85:15; 40 minutes: A:B=75:25; linear gradient. The retention timefor the pooled fraction was 8.5-11.5 minutes (for 3 minutes).

Yield: 56.3%. NMR (500 MHz, in D₂ O): δ^(TMS) =1.88 (3H, s, CH₃ onThymine), 1.94 (9H, s, CH₃ on Thymine), 2.27-2.60 (8H, m, 2'-CH₂ ×4),3.83 (1H, m, 5'-terminal 5'-CH₂ on dRib×1/2), 3.86 (1H, m, 5'-terminal5'-CH₂ on dRib×1/2), 4.12-4.27 and 4.36-4.46 (10H m, 4'-CH×4 and 5'-CH,on dRib×3), 4.56-4.62 (1H, m, 3'-terminal 3'-CH on dRib), 4.94-5.14 (3H,m, 3'-CH on dRib×3), 6.22-6.35 (4H, m, 1'-CH×4), 7.66 (1H, m, CH onThymine), 7.74-7.81 (3H, m, CH on Thymine×3)

(2) Synthesis of tri(ethoxy-β-galactose)-modified TetrathymidinePhosphorothioate Oligodeoxynucleotide

The galactose derivative of phosphoroamidite (Example 11) was dissolvedin acetonitrile to a concentration of 70 mM, and the solution wasimmediately applied onto an automated DNA synthesizer. It was thenreacted with tetrathymidine phosphorothioate oligodeoxynucleotidepreviously synthesized on a column for synthesis (15 μmol synthesisscale) according to the β-cyanoethylphosphoroamidite method. Amongsynthesizing reagents to be used in the automated DNA synthesizer, theoxidizing solution was replaced by Beaucage reagent for thio conversion.After completion of the reaction, a white amorphous powder was obtainedin the same manner as described in Example 15 (1).

Conditions for HPLC were the same as described in (1). The retentiontime for the pooled fraction was 15.0-21.0 minutes (for 6 minutes).

Yield: 28.5%. NMR (500 MHz, in D₂ O): δ^(TMS) =1.08-1.25 (10H m,--(CH₂)₅ -- on decanoyl), 1.48-1.62 (4H, m, --(CH₂)₂ -- on decanoyl),1.92-2.02 (14H, m, CH₃ on Thymine×4 and βCH₂ on Glu), 2.05-2.17 (2H, m,βCH₂ on Glu, -CH₂ CO on decanoyl), 2.20-2.60 (14H, m, --CH₂ CO ondecanoyl, 2'-CH₂ on dRib×4 and γCH₂ on Glu×2), 3.38-3.57 (6H, m, NCH₂ onethylene glycol×3), 3.54 (3H, dd, J=9 and 9 Hz, 2"-CH on Gal×3),3.64-4.02 (18H, m, OCH₂ on ethylene glycol×3, and 3"-CH, 5"-CH, 6"-CH₂on Gal×3), 3.93 (3H, m, 4"-CH on Gal×3), 4.12-4.28 and 4.42-4.50 (12H,m, 4'-CH and 5'-CH₂ on dRib×4), 4.25-4.30 (2H, m, αCH on Glu=2), 4.41(3H, m, 1"-CH on Gal×3), 4.57-4.63 (4H, m, 3'-CH on 3'-terminal dRib),5.01-5.28 (3H, m, 3'-CH on dRib×3), 6.27-6.38 (4H, m, 1'-CH on dRib×4),7.75-7.88 (4H, m, J=1 Hz, CH on Thymine×4)

Example 18

Synthesis of Phosphorothioate Oligodeoxynucleotide Derivative (2)

(1) Synthesis of Phosphorothioate Oligodeoxynucleotide

Using a 1 μmol synthesis scale column, a thio oligonucleotide having abase sequence of 5'-ATGCCCCTCAACGTT-3' (SEQ ID NO.1) was synthesized inthe same manner as described in Example 17 (1). The reaction program wascompleted leaving the DMT group at the 5' terminal intact. The columnwas washed with 3 ml of purified water, the carrier was removed from thecolumn and allowed to stand at room temperature for 24 hours afteradding 2 ml of concentrated aqueous ammonia (25%). After removing thecarrier by decantation, 0.4 ml of the reaction mixture was mixed withthe same volume of purified water, and then applied on an Olig-Pakcolumn (a product of Milipore) equilibrated with 1 M TEAA (pH 7.0). Thecolumn was washed with 3% aqueous ammonia (5 ml×3), purified water (5ml×3) and then with 5 ml of 2% trifluoroacetic acid to remove the DMTgroup at the 5' terminal. The column was then washed with purified water(5 ml×2) and then eluted with 40% acetonitrile. The abovementionedcolumn treatment was repeated 5 times. A pooled eluate fraction wasdried under vacuum to obtain a white amorphous powder.

Yield: 48.2%.

(2) Synthesis of tri(triethoxy-β-galactose)-modified ThioOligonucleotide

Using a 1 μmol synthesis scale column, tri(ethoxy-β-galactose)-modifiedphosphorothioate oligodeoxynucleotide having a base sequence of5'-ATGCCCCTCAACGTT-3' (SEQ ID NO.1) was synthesized in the same manneras described in Example 17 (2), but using phosphoroamidite of agalactose derivative (Example 12) in the modification reaction. Aftercompletion of the reaction, the column was washed with 3 ml of purifiedwater, the carrier was removed from the column and allowed to stand atroom temperature for 24 hours after adding 2 ml of concentrated aqueousammonia (25%). After removing the carrier by decantation, the reactionmixture was gel-filtrated on a Sephadex G-25 column (NAPS-25 column, aproduct of Pharmacia; bed volume: 9 ml) equilibrated with 100 mMphosphate buffer (pH 7.4) and the solvents were replaced by phosphatebuffer. The first eluate (3 ml) was discarded and the following eluate(3 ml) was collected to obtain a crude fraction.

In order to isolate a nucleotide with a bonded galactose derivative, anagarose column (bed volume: 10 ml, equilibrated with 100 mM phosphatebuffer) immobilized with a galactose bondable lectin, RCA 120, was used.The crude fraction was applied on the abovementioned column and anunbound fraction was eluted with 20 ml of 100 mM phosphate buffer. Next,a lectin-bound fraction was eluted with 20 ml of 100 mM phosphate buffercontaining 0.2 M galactose and collected. In order to remove galactosein the fraction, the following procedure was carried out using an ODScolumn (Sep-Pak Plus).

One tenth volume of 1 M TEAA was added to the lectin-bound fraction, andthe admixture was applied on an ODS column (Sep-Pak Plus) equilibratedwith 100 mM TEAA containing 10% acetonitrile. The ODS column was washedwith 10 ml of the buffer used for equilibration, and then further with10 ml of purified water. Next, elution was carried out with 70%acetonitrile and the eluate was dried under vacuum to obtain a whiteamorphous powder.

Yield: 42.8%.

Example 19

Synthesis of Phosphorothioate Oligodeoxynucleotide Derivative (3)

(1) Synthesis of Phosphorothioate Oligodeoxynucleotide

Using a 1 μmol synthesis scale column, a thio oligonucleotide having abase sequence of 5'-AACGTTGAGGGGCAT-3' (SEQ ID NO.2) was synthesized inthe same manner as described in Example 18 (1). A white amorphous powderwas obtained.

Yield: 65.3%.

(2) Synthesis of tri(triethoxy-β-galactose)-modified ThioOligonucleotide

Using a 1 μmol synthesis scale column,tri(triethoxy-β-galactose)-modified thio phosphorothioateoligodeoxynucleotide having a base sequence of 5'-AACGTTGAGGGGCAT-3'(SEQ ID NO.2) was synthesized in the same manner as described in Example18 (2). Phosphoroamidite (Example 12) was used in the modificationreaction. A white amorphous powder was obtained.

Yield: 31.5%.

Example 20

Synthesis of Phosphorothioate Oligodeoxynucleotide Derivative (4)

(1) Synthesis of Phosphorothioate Oligodeoxynucleotide

Using a 1 μmol synthesis scale column, a thio oligonucleotide having abase sequence of 5'-GGACTCAGACTCGCGTCC-3' (SEQ ID NO.3) was synthesizedin the same manner as described in Example 18 (1). A white amorphouspowder was obtained.

Yield: 62.8%.

(2) Synthesis of tri(triethoxy-β-galactose)-modified ThioOligonucleotide

Using a 15 μmol synthesis scale column,tri(triethoxy-β-galactose)-modified phosphorothioateoligodeoxynucleotide having a base sequence of 5'-GGACTCAGACTCGCGTCC-3'(SEQ ID NO.3) was synthesized in the same manner as described in Example17 (2), but using phosphoroamidite of a galactose derivative (Example12) in the modification reaction. After completion of the reaction, thecolumn was washed with 10 ml of purified water, the carrier was removedfrom the column and allowed to stand at room temperature for 24 hoursafter adding 10 ml of concentrated aqueous ammonia (25%). After removingthe carrier by decantation, 2 ml of the reaction mixture weregel-filtrated on an NAPS-25 column equilibrated with 100 mM phosphatebuffer, and the solvents were replaced by phosphate buffer. The firsteluate (3 ml) was discarded and the following eluate (3 ml) wascollected. The abovementioned gel filtration was repeated 5 times toobtain a crude fraction.

The crude fraction was applied on an agarose column (bed volume: 100 ml,equilibrated with 100 mM phosphate buffer) immobilized with a galactosebondable lectin, RCA 120, and an unbound fraction was removed by elutingwith 300 ml of 100 mM phosphate buffer. Next, a lectin-bound fractionwas eluted with 300 ml of 100 mM phosphate buffer containing 0.2 Mgalactose and collected. One tenth volume of 1 M TEAA was added, and theadmixture was applied on an ODS column (Sep-Pak Plus) equilibrated with100 mM TEAA containing 10% acetonitrile. The ODS column was washed with10 ml of the buffer used for equilibration, and then further with 10 mlof purified water. Next, elution was carried out with 70% acetonitrile,and the eluate was dried under vacuum to obtain a white amorphouspowder.

Yield: 24.6%.

Compounds of Examples 18 to 20 were analyzed using HPLC to confirm thepresence of a single peak only:

[Conditions for HPLC]

Column: Cation exchange column (Waters Gen-Pak Fax, 100×4.6 mm)

Column temperature: 80° C.

Detection: OD at 260 nm

Flow rate: 1.5 ml/min

Sample volume: 20 μl (equivalent to 1 OD₂₆₀)

Moving phase A: 100 mM Tris-HCl (pH 7.0)/30% acetonitrile

Moving phase B: 100 mM Tris-HCl (pH 7.0)/30% acetonitrile/2 M KBr

0 minute: A:B=95:5; 30 minutes: A:B=50:50 (0.1 M→1.0 M KBr lineargradient)

Example 21

Synthesis of Phosphorothioate Oligodeoxynucleotide Derivative (5)

Synthesis of di(triethoxy-β-galactose)-modified Thio Oligonucleotide

Using a 1 μmol synthesis scale column,di(triethoxy-β-galactose)-modified phosphorothioate oligodeoxynucleotidehaving a base sequence of 5'-AACGTTGAGGGGCAT-3' (SEQ ID NO.2) wassynthesized in the same manner as described in Example 18 (2). Agalactose derivative, phosphoroamidite (Example 14), was used in themodification reaction. A white amorphous powder was obtained.

Yield: 21.2%.

Example 22

Synthesis of Phosphorothioate Oligodeoxynucleotide Derivative (6)

Synthesis of triethoxy-β-galactose-modified PhosphorothioateOligodeoxynucleotide

Using a 1 μmol synthesis scale column, triethoxy-β-galactose-modifiedthio oligonucleotide having a base sequence of 5'-AACGTTGAGGGGCAT-3'(SEQ ID NO.2) was synthesized in the same manner as described in Example18 (2). A galactose derivative, phosphoroamidite (Reference Example 2),was used in the modification reaction. A white amorphous powder wasobtained.

Yield: 6.23%.

Protein Expression Suppression Test (1)

HepG2 cells were inoculated on a 6-well microplate (1×10⁶ per well) andincubated for 5 days to get nearly confluent growth. Each well waswashed with fresh RPMI1640 medium, after which 2 ml of the same mediumwas added. Further, 50 μl of PBS(-) containing the compounds of thepresent invention at various concentrations were added, and incubationwas carried out for 24 hours. Next, wells were washed with RPMI1640medium with no L-methionine, after which 30 μCi of [³⁵ S] L-methionineper well were added, and incubation was carried out for 20 hours. Afterwashing with ice-cold PBS(-), the cells were scraped using a scraper andcollected by centrifugation.

The following procedures were all conducted at 4° C. After addition of0.2 ml of lysis buffer (20 mM Tris-HCl (pH 7.4)/1 mM EDTA/1 mM PMSF/0.3%Nonidet P-40), the collected cells were dissolved by pipetting, and thesupernatant was obtained by centrifugation. After addition of 0.3 ml of20 mM Tris-HCl (pH 7.4)/150 mM NaCl and 1 pg of monoclonal human c-mycantibody (Clone 9E10, Catalogue # OP10, a product of Oncogene Science),the supernatant was allowed to stand in ice for 1 hour. Next, 0.1 ml ofProtein A-Sepharose gel was added and the admixture was shaken for 1hour. The gel was then washed 3 times with 1 ml of 20 mM Tris-HCL (pH7.4)/150 mM NaCl. After the addition of 100 μl of 0.1 M citric acid (pH2.0), the gel was allowed to stand for 10 minutes. Next, 50 μl of theresultant supernatant were mixed with the same volume of SDS-PAGE samplebuffer and heated in a boiling water bath for 3 minutes to obtain asample for SDS-PAGE. The sample (10 μl per well) was applied on a 8%SDS-PAGE (a product of TEFCO) and electrophoresis was carried out at 40mA for 1 hour. After fixing with 7% acetic acid and 20% methanol, thegel was dried at 60° C. for 1 hour. [³⁵ S] c-myc protein band wasdetected by exposing the gel to X-ray film (Hyperfilm β max, a productof Amersham).

Results are shown in FIG. 1. When the compounds of Examples 19 (1), 19(2), 21 and 22 were added to the medium at a concentration of 1 μM,there was no difference in the amount of expressed c-myc protein betweenthe compound of Example 19 (1) having no galactose residue and thecompound of Example 22 having 1 galactose residue. On the other hand,expression of c-myc protein was suppressed as the number of galactoseresidues increased in the compounds of Examples 22 and 19 (2). When thecompound of Example 19 (2) having 3 galactose residues and theunmodified compound of Example 19 (1) were added at a concentrationbetween 0.04 to 1.0 μM, suppression of synthesis was not observed withthe compound of Example 19 (1), but concentration-depending suppressionof synthesis was observed with the compound of Example 19 (2) (FIG. 2).

Protein Expression Suppression Test (2)

Suppression of expression of epidermal cell growth factor receptorprotein was tested as follows:

(1) Preparation of Rat Culture Hepatocytes

Under anesthesia with Nembutal, cannulas were inserted into the portalvein of male Wister rats (6 weeks of age). Perfusion from the portalvein was carried out using Hanks' buffer (containing 0.2 g/L EGTA, Ca²⁺,or Mg²⁺ free) heated to 37° C. at a flow rate of 20 ml/min for 10minutes to bleed the liver. Next, perfusion was carried out using Hanks'buffer containing 0.5 g/L collagenase for 10 minutes, after which theliver was shaken in ice-cooled Eagle's Minimum Essential Medium todisperse the cells.

After filtration through gauze, hepatocytes were purified bycentrifugation and that survivability was more than 90% was confirmed byTrypan Blue staining. The hepatocytes were suspended in Dulbecco'sMinimum Essential Medium containing 10% fetal calf serum, and the cellsuspension was inoculated into a collagen-coated 6-well microplate at10,000,000 cells/well. The plate was incubated in a 5% C0₂ atmosphere at37° C. for 2 hours. After washing the wells with medium to removeunabsorbed cells, fresh medium was added, and incubation was furthercontinued for 22 hours.

(2) Induction of Epidermal Growth Factor Receptor (EGFR) Down Regulation

Each well was washed with a test medium (Williams' Medium E containing10 nM insulin, 10 nM dexamethasone and 0.5% bovine serum albumin), and1.5 ml of the test medium was added. 100 μl of epidermal growth factor(10 μg/ml) were added to each well, and incubation was carried out for 6hours.

(3) Addition of Oligodeoxynucleotide

After washing with the test medium, the test medium containing thecompounds of Example 20 (1) or (2) was added at various concentrations,and incubation was carried out for 18 hours.

(4) Measurement of Specific Uptake of EGF via EGFR

The wells were washed with the test medium, fresh test medium was addedto the wells, then 0.1 mCi of [¹²⁵ I] EGF (a product of Amersham) wasadded to each well, and incubation was carried out for 1 hour. 1 ml of 1N NaOH was added to each well to dissolve the cells, after which a 0.1ml portion was sampled to determine the protein concentration. Anotherportion of 0.8 ml was sampled to measure radio activity to calculate theEGF uptake per unit hepatocyte protein. At the same time, [¹²⁵ I] EGFuptake with the addition of an excessive amount of unlabeled EGF (1.5μg/well) was measured. The specific EGF uptake via EGF receptor wascalculated by subtracting this value.

(5) Results

The amounts of EGF-specific binding in rat culture hepatocytes treatedby different procedures are shown in FIG. 3. "Negative control" meansthat step (4) was carried out after step (2) omitting step (3), in whichthe value represents the EGF-specific bonding immediately after EGFRdown regulation was induced. "Positive control" means that the amount ofEGF-specific binding was obtained by the same procedure, but with noaddition of EGF. "+EGF, Non AON" means that step (3) was carried outusing the test medium without the compound of Example 20 (1) or (2), and"Non-treatment" means that steps (2) and (3) were carried out with noaddition of EGF nor the compound of Example 20 (1) or (2), in which thelevel of binding was about the same as that of the positive control.When step (3) was carried out with the addition of the compound ofExample 20 (1) in the indicated concentrations, the amounts of bindingat the concentrations of 0.316 μM or less were about the same as thatfor the positive control. When step (3) was carried out with addition ofthe compound of Example 20 (2) at the indicated concentrations, theamount of EGF-specific binding decreased depending on the amounts ofcompound.

Growth Suppression Test

HepG2 cells were suspended in RPMI1640 medium containing 10% fetal calfserum and inoculated on a 96-well microplate (1000 cells per well), andthe plate was incubated at 37° C. for 2 days under a 5% CO₂ atmosphere.Each well was washed with Dulbecco's phosphate-buffered saline withCa²⁺, or Mg²⁺ free (PBS(-)), and then 100 μl of DM-160 medium containing0.5% BSA and the compounds of the present invention at variousconcentrations were added to each well. After incubation for 48 hours,the wells were washed again with PBS (-), the free medium containing ofthe compounds of the same concentrations and fresh medium were added toeach well, and incubation was carried out for another 48 hours. Eachwell was washed with PBS(-), 100 μl of Dulbecco's Minimum EssentialMedium without phenol red and 10 μl of a cell counting reagent (aproduct of Wako Pure Chemicals) were added, and then incubation wascarried out for 3 hours. Absorption at 450 nm was measured for each wellusing a microplate reader to calculate the number of viable cells.Results are shown in FIG. 4.

The compounds of Examples 19 (1) and 18 (1) had no effect in cell growthsuppression at all given concentrations. In contrast, the compounds ofExamples 19 (2) and 18 (2) in which a derivative having 3 galactoseresidues showed an improvement in suppressing cell growth. Inparticular, the compound of Example 19 (2) inhibited the growth at aconcentration of about 1 μM, in which suppressive activity was enhancedat least 7 times by the introduction of the galactose derivative.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES:  3                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  15 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE: Synthetic DNA                                     - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: phosphorotio - #ate                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - ATGCCCCTCA ACGTT              - #                  - #                      - #    15                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  15 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  Synthetic DNA                                    - -     (iv) ANTI-SENSE: YES                                                  - -     (ix) FEATURE:                                                                  (A) NAME/KEY:phosphorotioate                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - AACGTTGAGG GGCAT              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  18 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  Synthetic DNA                                    - -     (iv) ANTI-SENSE: YES                                                  - -     (ix) FEATURE:                                                                  (A) NAME/KEY:phosphorotioate                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - GGACTCAGAC TCGCGTCC             - #                  - #                      - #  18                                                                 __________________________________________________________________________

We claim:
 1. A compound of general formula (I): ##STR17## in which X isgroup (II): ##STR18## (in which Y is a leaving group) or group (III):##STR19## (in which Z is an oligonucleotide or a nucleotide derivativein which one or two of the oxygen atoms at a phosphoric ester bondingsite are substituted by other atoms or groups as shown by the followingformula: ##STR20## wherein A¹ and A² are selected from the followingcombinations:

    ______________________________________                                        A.sup.1        A.sup.2                                                        ______________________________________                                        --OH           ═O                                                           ═O --CH.sub.3                                                             --OH ═S                                                                   --SH ═S                                                                   ═O --O-Alkyl                                                              ═S --CH.sub.3                                                             ═O --NH-Alkyl                                                             ═O --BH.sub.3 ),                                                        ______________________________________                                    

T¹ is --(CH₂)s-- (in which s represents an integer between 2 and 10), or(CH₂ CH₂ O)t--(CH₂)₂ -- (in which t represents an integer between 1 and3), T² is --(CH₂)u-- (in which u represents an integer between 2 and10), --(CH₂ CH₂ O)v--(CH₂)₂ --(in which v represents an integer between1 and 3), or group (IV): ##STR21## (in which T¹ * and T¹ ** are each asdefined above for T¹, and n*, p*, q*, T³ *, T⁴ * and F³ are each asdefined below for n, p, q, T³, T⁴ and F¹, where each group and itsasterisk-labeled counterpart can be the same or different),T³, T⁴ andT⁵, which may be the same or different, each represent --CONH--,--NHCO-- or --O--, provided that when either one of T³, T⁴ and T⁵represents --O--, other two groups represent a group other than --O--,F¹ and F², which may be the same or different, each represent amonosaccharide selected from the group consisting of galactose, glucoseand galactosamine, or a derivative thereof, or a disaccharide consistingof the monosaccharide and/or the derivative thereof, wherein a hydroxylgroup(s) which does not participate in any reactions in themonosaccharide, the derivative thereof and the disaccharide can beprotected, and a hydroxyl (group(s) which does not participate in anyreactions in the monosaccharide, the derivative thereof, and thedisaccharide can be protected, m represents an integer between 0 and 10,n represents an integer between 0 and 4, p represents an integer between0 and 4, q represents an integer between 0 and 4 and r represents aninteger 0 or
 1. 2. The compound according to claim 1,wherein srepresents an integer between 2 and 8, t represents 2, v represents 2,T³, T⁴ and T⁵ represent --CONH--, F¹ and F², which may be the same ordifferent, each represent galactose, galactosamine,N-acetylgalactosamine, lactose, lactosamine or N-acetyllactosamine, mrepresents an integer 0 or between 2 and 10, n represents an integer 0,1 or
 2. P represents an integer 0, 1 or 2, q represents an integer 0, 1or 2, and r represents an integer
 1. 3. A compound of general formula(1a): ##STR22## in which X is group (II): ##STR23## (in which Y is aleaving group) or group (III) ##STR24## (in which z is anoligonucleotide or a nucleotide derivative in which one or two of theoxygen atoms at a phosphoric ester bonding site are substituted by otheratoms or groups as shown by the following formula: ##STR25## wherein A¹and A² are selected from the following combinations:

    ______________________________________                                        A.sup.1        A.sup.2                                                        ______________________________________                                        --OH           ═O                                                           ═O --CH.sub.3                                                             --OH ═S                                                                   --SH ═S                                                                   ═O --O-Alkyl                                                              ═S --CH.sub.3                                                             ═O --NH-Alkyl                                                             ═O --BH.sub.3 ),                                                        ______________________________________                                    

T¹ is --(CH₂)s-- (in which s represents an integer between 2 and 8), or--(CH₂ CH₂ O)₂ --(CH₂)₂ --, T² is --(CH₂)u-- (in which u represents aninteger between 2 and 8), --(CH₂ CH₂ O)₂ --(CH₂)--, or group (IVa):##STR26## (in which T¹ * and T¹ ** are as defined for T¹, and F³ is asdefined thereinafter for F¹, but can be the same as or different from T¹and F³ respectively),F¹ and F², which may be the same or different, eachrepresent a monosaccharide selected from the group consisting ofgalactose and galactosamine, or a derivative thereof, or a disaccharideconsisting of the monosaccharide and/or the derivative thereof, whereina hydroxyl group(s) which does not participate in any reactions in themonosaccharide, the derivative thereof and the disaccharide can beprotected, and m is an integer between 3 and
 9. 4. The compoundaccording to claim 3, wherein F¹ and F², which may be the same ordifferent, each represent galactose, galactosamine,N-acetylgalactosamine, lactose, lactosamine or N-acetyllactosamine. 5.The compound according to any one of claims 1 to 4, wherein X representsgroup (II).
 6. The compound according to claim 5, wherein Y representsan diisopropylamino group or a morpholyl group.
 7. The compoundaccording to any one of claims 1 to 4, wherein X represents group (III).8. The compound according to claim 7, wherein Z is selected from thegroup consisting of an oligodeoxyribonucleotide and anoligoribonucleotide and their phosphorothioate derivatives andmethylphosphate derivatives.
 9. The compound according to claim 8,wherein Z is an antisense oligonucleotide.
 10. The compound according toclaim 8, wherein Z is selected from the sequences consisting of SEQ IDNos: 1,2 and
 3. 11. A pharmaceutical composition comprising the compoundaccording to claim
 1. 12. The pharmaceutical composition according toclaim 11, which is used for a medicament selected from the groupconsisting of a therapeutic agent for a malignant tumor, an anti-viralagent, an antirheumatic agent, an anti-inflammatory agent, ananti-allergic agent, an immunosuppressive agent, a circulatory functionimproving agent and an endocrine function improving agent.
 13. A methodfor treating a disorder selected from the group consisting of amalignant tumor, a viral infection, an inflammatory disorder, anallergic disorder, an immune disorder, a circulatory disorder and anendocrine disorder, comprising administrating to an animal including ahuman the compound according to claim
 1. 14. A method for manufacturinga medicament selected from the group consisting of a therapeutic agentfor a malignant tumor, an anti-viral agent, an antirheumatic agent, ananti-inflammatory agent, an anti-allergic agent, an immunosuppressiveagent, a circulatory function improving agent and an endocrine functionimproving agent, which comprises admixing the compound of claim 1 with acarrier therefor.
 15. A medicament selected from the group consisting ofa therapeutic agent for a malignant tumor, an anti-viral agent, anantirheumatic agent, an anti-inflammatory agent, an anti-allergic agent,an immunosuppressive agent, a circulatory function improving agent andan endocrine function improving agent, which comprises the compound ofclaim 1 and a carrier therefor.